Inhibitors of histone deacetylase

ABSTRACT

The present invention relates to compounds of formula (I): 
     
       
         
         
             
             
         
       
     
     or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, wherein U, J, V, X, R 2a , R 2b , R 2c , R 5  and t are as described herein. The present invention relates generally to inhibitors of histone deacetylase and to methods of making and using them. These compounds are useful for promoting cognitive function and enhancing learning and memory formation. In addition, these compounds are useful for treating, alleviating, and/or preventing various conditions, including for example, neurological disorders, memory and cognitive function disorders/impairments, extinction learning disorders, fungal diseases and infections, inflammatory diseases, hematological diseases, and neoplastic diseases in humans and animals.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of and claims priority under 35U.S.C. §120 to U.S. patent application U.S. Ser. No. 14/065,276, filedOct. 28, 2013, which is a continuation of International PCT ApplicationNo. PCT/US2012/035814, filed on Apr. 30, 2012, which claims priority toand the benefit of U.S. Provisional Application No. 61/480,133, filed onApr. 28, 2011, the entire contents of each of which are incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates generally to inhibitors of histonedeacetylase and to methods of making and using them. These compounds areuseful for promoting cognitive function and enhancing learning andmemory formation. These compounds are useful for treating, alleviating,and/or preventing various conditions, including for example,neurological disorders, memory and cognitive functiondisorders/impairments, extinction learning disorders, fungal diseasesand infections, inflammatory diseases, hematological diseases, andneoplastic diseases in humans and animals.

BACKGROUND OF THE INVENTION

Inhibitors of histone deacetylases (HDAC) have been shown to modulatetranscription and to induce cell growth arrest, differentiation andapoptosis. HDAC inhibitors also enhance the cytotoxic effects oftherapeutic agents used in cancer treatment, including radiation andchemotherapeutic drugs. Marks, P., Rifkind, R. A., Richon, V. M.,Breslow, R., Miller, T., Kelly, W. K. Histone deacetylases and cancer:causes and therapies. Nat Rev Cancer, 1, 194-202, (2001); and Marks, P.A., Richon, V. M., Miller, T., Kelly, W. K. Histone deacetylaseinhibitors. Adv Cancer Res, 91, 137-168, (2004). Moreover, recentevidence indicates that transcriptional dysregulation may contribute tothe molecular pathogenesis of certain neurodegenerative disorders, suchas Huntington's disease, spinal muscular atrophy, amyotropic lateralsclerosis, and ischemia. Langley, B., Gensert, J. M., Beal, M. F.,Ratan, R. R. Remodeling chromatin and stress resistance in the centralnervous system: histone deacetylase inhibitors as novel and broadlyeffective neuroprotective agents. Curr Drug Targets CNS Neurol Disord,4, 41-50, (2005). A recent review has summarized the evidence thataberrant histone acetyltransferase (HAT) and histone deacetylases (HDAC)activity may represent a common underlying mechanism contributing toneurodegeneration. Moreover, using a mouse model of depression, Nestlerhas recently highlighted the therapeutic potential of histonedeacetylation inhibitors (HDAC5) in depression. Tsankova, N. M., Berton,O., Renthal, W., Kumar, A., Neve, R. L., Nestler, E. J. Sustainedhippocampal chromatin regulation in a mouse model of depression andantidepressant action. Nat Neurosci, 9, 519-525, (2006).

There are 18 known human histone deacetylases, grouped into four classesbased on the structure of their accessory domains. Class I includesHDAC1, HDAC2, HDAC3, and HDAC8 and have homology to yeast RPD3. HDAC4,HDAC5, HDAC7, and HDAC9 belong to class IIa and have homology to yeast.HDAC6 and HDAC10 contain two catalytic sites and are classified as classIIb. Class III (the sirtuins) includes SIRT1, SIRT2, SIRT3, SIRT4,SIRT5, SIRT6, and SIRT7. HDAC11 is another recently identified member ofthe HDAC family and has conserved residues in its catalytic center thatare shared by both class I and class II deacetylases and is sometimesplaced in class IV.

There is still much to be understood about the family of HDACs,including the varying functions of different HDACs and the range of HDACsubstrates. In order to learn more about the role that the individualHDACs play, it is important to develop compounds showing selectivity forindividual isoforms or small subsets of these isoforms. While somedegree of isoform selectivity has been shown by a few compounds, thisproblem of identifying selective inhibitors is far from solved, and theproblem is complicated by the interactions of the HDACs with each otheras well as other proteins (cofactors) that can possibly alter theirinteraction with various inhibitors (Glaser, et al., Biochem. Biophys.Res. Commun., 325, 683-690 (2004). Clinically, the optimal dose, timingand duration of therapy, as well as the most appropriate agents tocombine with HDAC inhibitors, are also still to be defined.

The findings to date suggest that HDAC inhibitors have great therapeuticpotential in promoting cognitive function, enhancing learning andmemory, and treating disease. There is a need to identifyspecific/selective HDAC inhibitors and to identify the structuralfeatures required for potent HDAC inhibitory activity.

SUMMARY OF THE INVENTION

The present invention provides compounds useful for the inhibition ofhistone deacetylase (HDAC). The invention provides a compound having theformula I:

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof. In formula I, the variables U, J, V, X, R^(1a), R^(1b), R^(2c),R⁵ and t can be selected from the respective groups of chemical moietieslater defined in the detailed description.

In addition, the invention provides pharmaceutical compositionscomprising an effective amount of a compound of the invention and apharmaceutical carrier, diluent, or excipient.

In one aspect, the invention provides a method of treating, alleviating,and/or preventing a condition in a subject comprising administering to asubject in need thereof an effective amount of a compound of theinvention or a pharmaceutically acceptable salt, hydrate, solvate orprodrug thereof. In one aspect, the condition is selected from aneurological disorder, memory or cognitive function disorder orimpairment, extinction learning disorder, fungal disease or infection,inflammatory disease, hematological disease, and neoplastic disease.

In one aspect, the invention provides a method of improving memory in anormal subject comprising administering to the subject in need thereofan effective amount of a compound of the invention or a pharmaceuticallyacceptable salt, hydrate, solvate, or prodrug thereof.

In one aspect, the invention provides a method of treating, alleviating,and/or preventing memory loss or impairment in a subject comprisingadministering to the subject in need thereof an effective amount of acompound of the invention or a pharmaceutically acceptable salt,hydrate, solvate, or prodrug thereof.

In one aspect, the invention provides a method of treating, alleviating,and/or preventing a cognitive function disorder or impairment in asubject in need thereof comprising administering to the subject aneffective amount of a compound of the invention or a pharmaceuticallyacceptable salt, hydrate, solvate, or prodrug thereof. In one aspect,the cognitive function disorder or impairment is associated withAlzheimer's disease, Huntington's disease, seizure induced memory loss,schizophrenia, Rubinstein Taybi syndrome, Rett Syndrome, Fragile X, Lewybody dementia, vascular dementia, ADHD, dyslexia, bipolar disorder andsocial, cognitive and learning disorders associated with autism,traumatic head injury, or attention deficit disorder. In one aspect, thecognitive function disorder or impairment is associated with an anxietydisorder, conditioned fear response, panic disorder, obsessivecompulsive disorder, post-traumatic stress disorder, phobia, socialanxiety disorder, substance dependence recovery or Age Associated MemoryImpairment (AAMI), or Age Related Cognitive Decline (ARCD).

In one aspect, the invention provides a method of treating, alleviating,and/or preventing an inflammatory disease in a subject in need thereofcomprising administering to the subject an effective amount of acompound or a pharmaceutically acceptable salt, hydrate, solvate, orprodrug thereof.

In one aspect, the invention provides a method of treating, alleviating,and/or preventing a fungal disease or infection in a subject in needthereof comprising administering to the subject an effective amount of acompound of the invention or a pharmaceutically acceptable salt,hydrate, solvate, or prodrug thereof.

In one aspect, the invention provides a method of treating, alleviating,and/or preventing a hematological disease in a subject comprisingadministering to the subject in need thereof an effective amount of acompound of the invention or a pharmaceutically acceptable salt,hydrate, solvate, or prodrug thereof. In one aspect, the hematologicaldisease is selected from acute myeloid leukemia, acute promyelocyticleukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia,myelodysplastic syndromes, and sickle cell anemia. In one aspect, thehematological disease is sickle cell anemia.

In one aspect, the invention provides a method of treating, alleviating,and/or preventing a neoplastic disease in a subject comprisingadministering to the subject in need thereof an effective amount of acompound of the invention or a pharmaceutically acceptable salt,hydrate, solvate, or prodrug thereof. In one aspect, the neoplasticdisease is cancer.

In one aspect, the invention provides a method of treating, alleviating,and/or preventing a psychiatric disease (depression, mood, maniadisorders etc.) in a subject comprising administering to the subject inneed thereof an effective amount of a compound of the invention or apharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof.

In one aspect, the invention provides a method, wherein the method is acombination therapy further comprising administering to the subject (1)a pharmaceutically active ingredient or exposing the subject to (2)cognitive behavioral therapy (CBT), (3) psychotherapy, (4) behavioralexposure treatments, (5) virtual reality exposure (VRE) or (6) cognitiveremediation therapy or (7) any combination thereof.

In one aspect, the invention provides a combination therapy fortreating, alleviating, and/or preventing post-traumatic stress disorder(PTSD) or Alzheimer's disease in a subject comprising administering tothe subject in need thereof an effective amount of (1) a compound of theinvention or a pharmaceutically acceptable salt, hydrate, solvate, orprodrug thereof and (2) a pharmaceutically active ingredientadministered selected from Aricept®, memantine, and galantamine.

In one aspect, the invention provides a method of treating extinctionlearning disorders in a subject in need thereof comprising administeringto the subject an effective amount of a compound of the invention or apharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof.In one aspect, the extinction learning disorder is

fear extinction deficit. In one aspect, the extinction learning disorderis post-traumatic stress disorder. In one aspect, the method is acombination therapy for treating extinction learning disorders in asubject in need thereof comprising administering to the subject (1) aneffective amount of a compound of the invention or a pharmaceuticallyacceptable salt, hydrate, solvate, or prodrug thereof and (2) exposingthe subject to cognitive behavioral therapy (CBT), psychotherapy,behavioral exposure treatments, virtual reality exposure (VRE) orcognitive remediation therapy.

In one aspect, the invention provides a method wherein the compound ofthe invention or a pharmaceutically acceptable salt, hydrate, solvate,or prodrug thereof is administered by a route selected from oral,parenteral, intramuscular, intranasal, sublingual, intratracheal,inhalation, ocular, vaginal, rectal, and intracerebroventricular.

In one aspect, the invention provides a method, wherein the subject is ahuman.

In one aspect, the invention provides a method of synthesizing acompound of the invention or a pharmaceutically acceptable salt,hydrate, solvate, or prodrug thereof.

In one aspect, the invention provides a kit containing one or morecompounds of the invention or a pharmaceutically acceptable salt,hydrate, solvate, or prodrug thereof. In one aspect, the kit furthercontains a pharmaceutically active ingredient.

In one aspect, the invention provides a method of increasing synapticdensity in a subject comprising administering to the subject in need ofsuch increase an effective amount of a compound of the invention or apharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof.In one aspect, the invention provides a method of increasing synapticplasticity in a subject comprising administering to the subject in needof such increase an effective amount of a compound of the invention or apharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof.In one aspect, the invention provides a method of increasing dendriticdensity in neurons in a subject comprising administering to the subjectin need of such increase an effective amount of a compound of theinvention or a pharmaceutically acceptable salt, hydrate, solvate, orprodrug thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1C are dose response curves of histone acetylation(AcH4K12) in primary neuronal cultures by compounds of the invention.FIGS. 1B and 1D are dose response curves of histone acetylation (AcH3K9)in primary neuronal cultures by compounds of the invention.

FIG. 2 is a heat map indicating changes in histone acetylation andmethylation states regulated by treatment of primary neuronal cultureswith compound 103 or compound A (positive control).

FIG. 3A is a Western blot showing detection of HDACs 1, 2 and 3 andknown members of endogenous HDAC2 complexes in immunoprecipitated HDAC2complexes. FIG. 3B is a bar chart indicating the enzymatic activity ofthe immunoprecipitated HDAC2 complexes from mouse forebrain. FIG. 3C isa graph indicating the % inhibition of enzymatic activity over time bycompounds of the invention or compound A (positive control).

FIG. 4 is a bar chart indicating increased freezing in a contextual fearconditioning paradigm following administration of compounds of theinvention or compound A (positive control; 10 mg/kg) in wild type mice.

FIG. 5 is a bar chart indicating increased freezing in a contextual fearconditioning paradigm following administration of compounds of theinvention or compound A (positive control; 1 mg/kg) in 6-week inducedCK-p25 mice (neurodegenerative mouse model).

FIG. 6A is a bar chart indicating the relative histone acetylation ofH3K9 and H4K12 in C57BL/6 mouse cortex following chronic administrationof compounds of the invention or compound A (positive control) comparedto untreated (vehicle) mice. FIG. 6B shows the representative Westernblots.

FIG. 7 is an immunoblot analysis of histone acetylation (AcH4K5 andAcH4K12) in CK-p25 mouse hippocampus following chronic administration ofcompounds of the invention or compound A (positive control).

FIG. 8A is a Western blot showing the decrease of GFAP protein afteradministration of compound 103. FIG. 8B is a bar chart indicating thelevel of GFAP normalized to GAPDH.

FIG. 9A is a line graph of the total locomotor activity over time (min)in C57BL/6 mice after chronic administration of compound 103 of theinvention in the amphetamine induced hyperactivity (AIH) mouse model.FIG. 9B is a bar chart of the total activity during the test period.

FIG. 10A is a graph of the total locomotor activity over time (min) inC57BL/6 mice following chronic administration of compound 191 of theinvention in AIH. FIG. 10B is a bar chart of the total activity duringthe test period.

FIG. 11 is a bar chart indicating that chronic administration ofcompound 191 in C57BL/6 mice decreases the immobility time during theforced swim test.

FIG. 12 is a bar chart indicating that chronic administration ofcompound 54 in C57BL/6 mice decreases the immobility time during theforced swim test.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compounds, pharmaceutical compositions andmethods for inhibiting class I histone deacetylase enzymatic activity.The invention also provides compounds, pharmaceutical compositions andmethods for promoting cognitive function and treating, alleviatingand/or preventing various conditions disease e.g., neurologicaldisorders, memory and cognitive function disorders/impairments,extinction learning disorders, fungal diseases, inflammatory diseases,hematological diseases, and neoplastic diseases. The patent andscientific literature referred to herein establishes knowledge that isavailable to those with skill in the art. The issued patents,applications, and references that are cited herein are herebyincorporated by reference to the same extent as if each was specificallyand individually indicated to be incorporated by reference. In the caseof inconsistencies, the present disclosure will prevail.

For purposes of the present invention, the following definitions will beused (unless expressly stated otherwise):

The general chemical terms used throughout have their usual meanings.For example, the term alkyl refers to a branched or unbranched saturatedhydrocarbon group. The term “n-alkyl” refers to an unbranched alkylgroup. The term “C_(x)-C_(y) alkyl” refers to an alkyl group havingbetween x and y carbon atoms, inclusively, in the branched or unbranchedhydrocarbon group. By way of illustration, but without limitation, theterm “C₁-C₈ alkyl” refers to a straight chain or branched hydrocarbonmoiety having from 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. “C₁-C₆”refers to a straight chain or branched hydrocarbon moiety having from 1,2, 3, 4, 5, or 6 carbon atoms. “C₁-C₄ alkyl” refers to a straight chainor branched hydrocarbon moiety having from 1, 2, 3, or 4 carbon atoms,including methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, and tert-butyl. The term “C₁-C₄ n-alkyl” refers to straightchain hydrocarbon moieties having from 1 to 4 carbon atoms includingmethyl, ethyl, n-propyl, and n-butyl. The term “C₃-C₆ cycloalkyl” refersto cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. The term “C₃-C₇cycloalkyl” also includes cycloheptyl. The term “C₃-C₈ cycloalkyl” alsoincludes cyclooctyl. Cycloalkylalkyl refers to cycloalkyl moietieslinked through an alkyl linker chain, as for example, but withoutlimitation, cyclopropylmethyl, cyclopropylethyl, cyclopropylpropyl,cyclopropylbutyl, cyclobutylmethyl, cyclobutylethyl, cyclobutylpropyl,cyclopentylmethyl, cyclopentylethyl, cyclopentylpropyl,cyclohexylmethyl, cyclohexylethyl, and cyclohexylpropyl. Each alkyl,cycloalkyl, and cycloalkylalkyl group may be optionally substituted asspecified herein.

The term “C₄-C₈ cycloalkenyl” refers to cyclobutenyl, cyclopentyl,cyclohexenyl, cycloheptenyl, and cyclooctenyl rings having one or moresites of unsaturation e.g., one or more double bonds.

The term “3 to 8 membered ring” includes a 3, 4, 5, 6, 7, and 8-memberedring.

The terms “alkoxy”, “phenyloxy”, “benzoxy” and “pyrimidinyloxy” refer toan alkyl group, phenyl group, benzyl group, or pyrimidinyl group,respectively, each optionally substituted, that is bonded through anoxygen atom.

The term “halogen” refers to fluoro, chloro, bromo, or iodo.

The term “hydroxyl” means OH.

The term “aryl” or “aromatic ring” alone or in combination, means acarbocyclic aromatic system containing one, two or three rings whereinsuch rings may be attached together in a pendent manner or may be fused.The term “aryl” or “aromatic ring” embraces aromatic radicals such asphenyl (e.g., C₆H₅—), naphthyl, tetrahydronapthyl, indane and biphenyl,and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, allof which may be optionally substituted.

The term “heteroaryl” or “heteroaromatic ring” as used herein includes5-, 6- and 7-membered single-ring aromatic groups that may include from1, 2, 3, or 4 heteroatoms, for example, pyrrole, furan, thiophene,imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine,pyridazine, azepine, oxepine, oxazine, triazine and pyrimidine, and thelike. Those aryl groups having heteroatoms in the ring structure mayalso be referred to as “aryl heterocycles” or “heteroaromatics.” Aheteroaryl or heteroaromatic ring can be monocyclic, bicyclic, ortricyclic, wherein such rings may be attached together in a pendentmanner or may be fused. In one aspect, the heteroaryl or heteroaromaticring is a 5-, 6-, or 7-membered single ring that includes from 1, 2, 3,or 4 heteroatoms. A heteroaryl or heteroaromatic ring can be substitutedat one or more ring positions with such substituents as described above,for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl,cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromaticor heteroaromatic moieties, —CF₃, —CN, or the like.

The term “heterocyclic ring” or “heterocycle” is taken to mean asaturated, unsaturated, or partially unsaturated containing from 1, 2,3, or 4 heteroatoms selected from nitrogen, oxygen and sulfur, said ringoptionally being benzofused. A heterocylic ring can be multicyclic e.g.,bicyclic or tricyclic. The term “3- to 8-membered heterocyclic ring”refers to a ring having from 3, 4, 5, 6, 7 or 8 atoms. The term “3- to6-membered heterocyclic ring” refers to a ring having from 3, 4, 5, or 6atoms. The term “5- to 6-membered heterocyclic ring” refers to a ringhaving 5 or 6 atoms. Exemplary heterocyclic rings, for the purposes ofthe present invention, include furanyl, thiophenyl (thienyl orthiopheneyl), pyrrolyl, pyrrolidinyl, pyridinyl, N-methylpyrrolyl,oxazolyl, isoxazolyl, pyrazolyl, imidazolyl, triazolyl, oxadiazolyl,thiadiazolyl, thiazolyl, thiazolidinyl, N-acetylthiazolidinyl,pyrimidinyl, pyrazinyl, pyridazinyl, and the like. Heterocyclic ringsinclude bicyclic rings for example, 3-azabicyclo[3.1.0]hexane,8-oxa-3-azabicyclo[3.2.1]octane. Benzofused heterocyclic rings includeisoquinolinyl, benzoxazolyl, benzodioxolyl, benzothiazolyl, quinolinyl,benzofuranyl, benzothiophenyl, indolyl, and the like, all of which maybe optionally substituted, which also of course includes optionallysubstituted on the benzo ring when the heterocycle is benzofused.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc. As used herein, the term “substituted” iscontemplated to include all permissible substituents of organiccompounds unless otherwise specified. In a broad aspect, the permissiblesubstituents include acyclic and cyclic, branched and unbranched,carbocyclic and heterocyclic, aromatic and nonaromatic substituents oforganic compounds. The permissible substituents can be one or more andthe same or different for appropriate organic compounds. For purposes ofthis invention, the heteroatoms such as nitrogen may have hydrogensubstituents and/or any permissible substituents of organic compoundsdescribed herein which satisfy the valences of the heteroatoms. Thisinvention is not intended to be limited in any manner by the permissiblesubstituents of organic compounds.

The term “pharmaceutical” or “pharmaceutically acceptable” when usedherein as an adjective, means substantially non-toxic and substantiallynon-deleterious to the recipient.

By “pharmaceutical formulation” it is further meant that the carrier,solvent, excipient(s) and salt must be compatible with the activeingredient of the formulation (e.g. a compound of the invention). It isunderstood by those of ordinary skill in this art that the terms“pharmaceutical formulation” and “pharmaceutical composition” aregenerally interchangeable, and they are so used for the purposes of thisapplication.

The term “acid addition salt” refers to a salt of a compound of theinvention prepared by reaction of a compound of the invention with amineral or organic acid. For exemplification of pharmaceuticallyacceptable acid addition salts see, e.g., Berge, S. M, Bighley, L. D.,and Monkhouse, D. C., J. Pharm. Sci., 66:1, 1977. Compounds of thisinvention which are amines, are basic in nature and accordingly reactwith any of a number of inorganic and organic acids to formpharmaceutically acceptable acid addition salts.

Pharmaceutically acceptable acid addition salts of the invention can beformed by reacting a compound of the invention with an equimolar orexcess amount of acid. Alternatively, hemi-salts can be formed byreacting a compound of the invention with the desired acid in a 2:1ratio, compound to acid. The reactants are generally combined in amutual solvent such as diethylether, tetrahydrofuran, methanol, ethanol,isopropanol, benzene, or the like. The salts normally precipitate out ofsolution within about one hour to about ten days and can be isolated byfiltration or other conventional methods.

Inorganic acids commonly employed to form such salts includehydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid,phosphoric acid, and the like. Organic acids commonly employed to formsuch salts include p-toluenesulfonic acid, methanesulfonic acid, oxalicacid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citricacid, benzoic acid, acetic acid and the like. Examples of suchpharmaceutically acceptable salts thus are the sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide,iodide, acetate, propionate, decanoate, caprylate, acrylate, formate,isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate,succinate, hemisuccinate, suberate, sebacate, fumarate, maleate,butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate,methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate,phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate,phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycollate,tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate,naphthalene-2-sulfonate, mandelate and the like.

Some of the compounds of the present invention may exist in unsolvatedas well as solvated forms such as, for example, hydrates.

Since prodrugs are known to enhance numerous desirable qualities ofpharmaceuticals (e.g., solubility, bioavailability, manufacturing, etc.)the compounds of the present invention can be delivered in prodrug form.Thus, the present invention is intended to cover prodrugs of thecompounds of the invention, methods of delivering the same andcompositions containing the same. “Prodrugs” are intended to include anycovalently bonded carriers that release an active parent drug of thepresent invention in vivo when such prodrug is administered to amammalian subject. Prodrugs are prepared by modifying functional groupspresent in the compound in such a way that the modifications arecleaved, either in routine manipulation or in vivo, to the parentcompound. Prodrugs include compounds of the invention wherein a hydroxylor amino, group is bonded to any group that, when the prodrug of thepresent invention is administered to a mammalian subject, it cleaves toform a free hydroxyl or free amino group, respectively. Examples ofprodrugs include, but are not limited to, acetate, formate, and benzoatederivatives of alcohol and amine functional groups in the compounds ofthe present invention.

“Solvate” means a solvent addition form that contains either astoichiometric or non stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate, when the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one of the substances in whichthe water retains its molecular state as H₂O, such combination beingable to form one or more hydrate.

The term “suitable solvent” refers to any solvent, or mixture ofsolvents, inert to the ongoing reaction that sufficiently solubilizesthe reactants to afford a medium within which to effect the desiredreaction.

The compounds described herein can have asymmetric centers. Compounds ofthe present invention containing an asymmetrically substituted atom canbe isolated in optically active or racemic forms. It is well known inthe art how to prepare optically active forms, such as by resolution ofracemic forms or by synthesis from optically active starting materials.Many geometric isomers of olefins, C═N double bonds, and the like canalso be present in the compounds described herein, and all such stableisomers are contemplated in the present invention. Cis and transgeometric isomers of the compounds of the present invention aredescribed and can be isolated as a mixture of isomers or as separateisomeric forms. All chiral, diastereomeric, racemic, and geometricisomeric forms of a structure are intended, unless specificstereochemistry or isomeric form is specifically indicated. Allprocesses used to prepare compounds of the present invention andintermediates made therein are considered to be part of the presentinvention. All tautomers of shown or described compounds are alsoconsidered to be part of the present invention. Furthermore, theinvention also includes metabolites of the compounds described herein.

The invention also comprehends isotopically-labeled compounds, which areidentical to those recited in the formulae of the invention, but for thefact that one or more atoms are replaced by an atom having an atomicmass or mass number different from the atomic mass or mass number mostcommonly found in nature. Examples of isotopes that can be incorporatedinto compounds of the invention include isotopes of hydrogen, carbon,nitrogen, fluorine, such as ³H, ¹¹C, ¹⁴C, ²H and ¹⁸F.

Compounds of the present invention and salts, hydrates, solvates orprodrugs of said compounds that contain the aforementioned isotopesand/or other isotopes of other atoms are within the scope of the presentinvention. Isotopically-labeled compounds of the present invention, forexample those into which radioactive isotopes such as ³H, ¹⁴C areincorporated, are useful in drug and/or substrate tissue distributionassays. Tritiated, i.e., 3H, and carbon-14, i.e., ¹⁴C, isotopes areparticularly preferred for their ease of preparation and detectability.¹¹C and ¹⁸F isotopes are particularly useful in PET (positron emissiontomography). PET is useful in brain imaging. Further, substitution withheavier isotopes such as deuterium, i.e., ²H, can afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life or reduced dosage requirements and,hence, may be preferred in some circumstances, isotopically labeledcompounds of this invention can generally be prepared by carrying outthe procedures disclosed in the Schemes and/or in the Examples below, bysubstituting a readily available isotopically labeled reagent for anon-isotopically labeled reagent. In one embodiment, the compounds ofthe invention, salts, hydrates, solvates, or prodrugs thereof are notisotopically labelled.

When any variable (e.g., R^(x)) occurs more than one time in anyconstituent or formula for a compound, its definition at each occurrenceis independent of its definition at every other occurrence. Thus, forexample, if a group is shown to be substituted with one or more R^(x)moieties, then R^(x) at each occurrence is selected independently fromthe definition of R^(x). Also, combinations of substituents and/orvariables are permissible, but only if such combinations result instable compounds within a designated atom's normal valency.

As used herein, the term “treat,” “treating,” “alleviate,” or“alleviating” herein, is meant decreasing the symptoms, markers, and/orany negative effects of a condition in any appreciable degree in apatient who currently has the condition. In some embodiments, treatmentmay be administered to a subject who exhibits only early signs of thecondition for the purpose of decreasing the risk of developing thedisease, disorder, and/or condition.

As used herein, the term “prevent,” “prevention,” or “preventing” refersto any method to partially or completely prevent or delay the onset ofone or more symptoms or features of a disease, disorder, and/orcondition. Prevention may be administered to a subject who does notexhibit signs of a disease, disorder, and/or condition.

As used herein, “subject” means a human or animal (in the case of ananimal, more typically a mammal). In one aspect, the subject is a human.Such subject can be considered to be in need of treatment with an HDACinhibitor.

As used herein, “unsaturated” refers to compounds or structures havingat least one degree of unsaturation (e.g., at least one double or triplebond).

As used herein, the term “Compound A” refers to the known compoundCI-994.

COMPOUNDS OF THE INVENTION

In one aspect, the invention relates to a compound of formula I:

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof, whereinU is selected from single bond, CR^(2e)R^(2f)—CR^(2g)R^(2h), NR^(2d),NR^(2d)—NR^(2d); and O;J is selected from NH₂, OH, and SH;V is selected from C and N, provided that when V is N, one of R^(2a),R^(2b), or R^(2c) is absent;X is selected from hydrogen, deuterium, methyl, CF₃, and halogen;R^(2a) is selected from hydrogen, halogen, OH, NH₂, and C₁-C₈ alkyl;R^(2b) is selected from hydrogen, halogen, OH, NH₂, and C₁-C₈ alkyl;R^(2c) is selected from hydrogen, halogen, OH, NH₂, and C₁-C₈ alkyl;R^(2d) is selected from NH₂, hydrogen, and C₁-C₈ alkyl;R^(2d′) is selected from NH₂, hydrogen, and C₁-C₈ alkyl;R^(2e), R^(2f), R^(2g), and R^(2h) are each independently selected fromhydrogen, halogen, and C₁-C₄ alkyl;or taken together two of R^(2a), R^(2b), and R^(2c) form ═O,or taken together two of R^(2a), R^(2b), and R^(2c) form a C₃-C₈cycloalkyl ring, C₄-C₈ cycloalkenyl ring, or 3 to 8 membered saturatedor partially unsaturated heterocyclic ring and the remaining R^(2a),R^(2b), or R^(2c) is absent or selected from hydrogen, halogen, OH, NH₂,and C₁-C₈ alkyl, further wherein said cycloalkyl, cycloalkenyl, andheterocyclic ring are unsubstituted or substituted with one or moreR^(x);or alternatively, taken together two of R^(2a), R^(2b), R^(2c) andR^(2d) form a C₃-C₈ cycloalkyl ring, C₄-C₈ cycloalkenyl ring, or 3 to 8membered saturated or partially unsaturated heterocyclic ring and theremaining R^(2a), R^(2b), or R^(2c) is selected from hydrogen, halogen,OH, NH₂, and C₁-C₈ alkyl or R^(2d) is hydrogen, NH₂, or C₁-C₈ alkyl,further wherein said cycloalkyl, cycloalkenyl, and heterocyclic ring areunsubstituted or substituted with one or more R^(x);or taken together two of R^(2a), R^(2b), and R^(2c) form an aromatic orheteroaromatic ring and the remaining R^(2a), R^(2b), or R^(2c) isabsent, provided that when two of R^(2a), R^(2b), and R^(2c) form anaromatic or heteroaromatic ring and the remaining R^(2a), R^(2b), orR^(2c) is absent, U is not a single bond when t is 0,further wherein said aromatic and heteroaromatic ring are unsubstitutedor substituted with one or more R^(x),or taken together R^(2e) and R^(2f) or R^(2g) and R^(2h) taken togetherform ═O;or taken together two of R^(2e), R^(2f), R^(2g), and R^(2h) on twoadjacent carbon atoms together with the bond between said adjacentcarbon atoms form a carbon-carbon double bond;or taken together two of R^(2e), R^(2f), R^(2g), and R^(2h) on twoadjacent carbon atoms together with the intervening atoms to which theyare attached form a 3 to 8 membered saturated or partially saturatedring;each R^(x) is independently selected from (CH₂)_(z)NH₂, (CH₂)_(z)NHR³,(CH₂)_(z)NR³R³, OR³, OCF₃, OCH₂F, OCHF₂, (CH₂)_(z)-aromatic ring,(CH₂)_(z)-heterocyclic ring, hydroxyl, halogen, C₁-C₈ alkyl, (C₁-C₈alkyl)CF₃, (C₁-C₈ alkyl)OH, C(O)R³, (CH₂)_(z)C(O)NH₂, (CH₂)_(z)C(O)NHR³,(CH₂)_(z)C(O)NR³R³, (CH₂)_(z)NHC(O)R⁴, and (CH₂)_(z)NR⁴C(O)R⁴;or taken together two R^(x) attached to the same carbon atom of acycloalkyl, cycloalkenyl or heterocyclic ring together form ═O;or taken together two R^(x) form a C₃-C₈ cycloalkyl ring, C₄-C₈cycloalkenyl ring or 3 to 8 membered saturated or partially unsaturatedheterocyclic ring, further wherein said cycloalkyl, cycloalkenyl, andheterocyclic ring are unsubstituted or substituted with one or moreR^(z); or taken together two R^(x) form an aromatic ring orheteroaromatic ring, further wherein said aromatic and heteroaromaticring are unsubstituted or substituted with one or more R^(z);each R^(z) is independently selected from halogen, C₁-C₄ alkyl, OH, OR³,CF₃, OCF₃, OCH₂F, OCHF₂, NH₂, NHR³, NR³R³, and C(O)CH₃;R³ is selected from C₁-C₈ alkyl and O(C₁-C₈ alkyl);R⁴ is selected from C₁-C₈ alkyl and CF₃;R⁵ is selected from hydrogen, deuterium, halogen, OH, OR⁶, CF₃, C₁-C₈alkyl, C₂-C₈ alkene, (CH₂)_(u)-5-6 membered saturated, unsaturated, orpartially unsaturated heterocyclic ring, (CH₂)_(v)—C₃-C₈ cycloalkylring, (C₁-C₈-alkyl)_(w)-C₄-C₅cycloalkenyl ring, (CH₂)_(s)-aromatic ring,and (CH₂)_(s)-heteroaromatic and wherein said heterocyclic, cycloalkyl,cycloalkenyl, heteroaromatic, andaromatic ring are unsubstituted or substituted with one or more R^(y)and said alkene is substituted with one or more R^(T);each R^(y) is independently selected from halogen, OR⁶, NH₂, NHR⁶,NR⁶R⁶, OH, CF₃, aromatic ring, C(O)R⁶, C₁-C₈ alkyl, C₂-C₈ alkenyl, andC₂-C₈ alkynyl;R⁶ is C₁-C₈ alkyl;each R^(T) is independently selected from hydrogen, halogen, Si(R³)₃,phenyl, and C₁-C₈ alkyl;u is selected from 0, 1, and 2;v is selected from 0, 1, and 2;w is selected from 0, 1, and 2;s is selected from 0, 1, and 2;t is selected from 0, 1, and 2, andz is selected from 0, 1, 2, and 3.

In one aspect, the invention relates to a compound having the formula Ior a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof, wherein U is selected from single bond,CR^(2e)R^(2f)—CR^(2g)R^(2h), NR^(2d), NR^(2d)—NR^(2d′) and O; J isselected from NH₂, OH, and SH; V is selected from C and N, provided thatwhen V is N, one of R^(2a), R^(2b), or R^(2c) is absent; X is selectedfrom hydrogen, methyl, CF₃, and halogen; R^(2a) is selected fromhydrogen, halogen, OH, NH₂, and C₁-C₈ alkyl; R^(2b) is selected fromhydrogen, halogen, OH, NH₂, and C₁-C₈ alkyl; R^(2c) is selected fromhydrogen, halogen, OH, NH₂, and C₁-C₈ alkyl; R^(2d) is selected fromNH₂, hydrogen, and C₁-C₈ alkyl; R^(2d′) is selected from NH₂, hydrogen,and C₁-C₈ alkyl; R^(2e), R^(2f), R^(2g) and R^(2h) are eachindependently selected from hydrogen, halogen, and C₁-C₄ alkyl; or takentogether two of R^(2a), R^(2b), and R^(2c) form ═O; or taken togethertwo of R^(2a), R^(2b), and R^(2c) form a C₃-C₈ cycloalkyl ring, C₄-C₈cycloalkenyl ring, or 3 to 8 membered saturated or partially unsaturatedheterocyclic ring, and the remaining R^(2a), R^(2b), or R^(2c) isselected from hydrogen, halogen, OH, NH₂, and C₁-C₈ alkyl; or takentogether two of R^(2a), R^(2b), and R^(2c) form an aromatic orheteroaromatic ring and the remaining R^(2a), R^(2b), or R^(2c) isabsent, provided that U is not single bond and t is not 0; further,wherein said cycloalkyl, cycloalkenyl, heterocyclic, aromatic andheteroaromatic ring (formed by two of R^(2a), R^(2b), and R^(2c)) areunsubstituted or substituted with one or more R^(x), or taken togethertwo of R^(2e), R^(2f), R^(2g), and R^(2h) on two adjacent carbon atomstogether with the bond between said adjacent carbon atoms form acarbon-carbon double bond; or taken together two of R^(2e), R^(2f),R^(2g), and R^(2h) on two adjacent carbon atoms together with theintervening atoms to which they are attached form a 3 to 8 memberedsaturated or partially saturated ring;

each R^(x) is independently selected from NH₂, NHR³, NR³R³, OR³,(CH₂)_(z)C₆H₆, hydroxyl, halogen, C₁-C₈ alkyl, (C₁-C₈)CF₃, (C₁-C₈)OH,C(O)R³ and (CH₂)_(z)NHC(O)R⁴,or taken together two R^(x) attached to the same carbon atom of acycloalkyl, cycloalkenyl or heterocyclic ring together form ═O; or takentogether two R^(x) form a C₃-C₈ cycloalkyl ring, C₄-C₈ cycloalkenyl ringor 3 to 8 membered saturated or partially unsaturated heterocyclic ring;further wherein said cycloalkyl, cycloalkenyl, and heterocyclic ring(formed by two R^(x)) are optionally substituted with one or more R^(z);each R^(z) is independently selected from halogen, C₁-C₄ alkyl, OH, NH₂,and C(O)CH₃; R³ is selected from C₁-C₈ alkyl and O(C₁-C₈ alkyl); R⁴ isselected from C₁-C₈ alkyl and CF₃; R⁵ is selected from hydrogen,halogen, OH, OR⁶, CF₃, CH₃, C₂₋₈ alkene, (CH₂)_(u)-5-6 memberedsaturated, unsaturated, or partially unsaturated heterocyclic ring,(CH₂)_(v)—C₃-C₈ cycloalkyl ring, (C₁-C₈-alkyl)_(w)-C₄-C₈ cycloalkenylring, (CH₂)_(s)-aromatic ring, and (CH₂)_(s)-heteroaromatic, and whereinsaid heterocyclic, cycloalkyl, cycloalkenyl, aromatic and heteroaromaticring are unsubstituted or substituted with one or more R^(y) and saidalkene is substituted with one or more R^(T); each R^(y) isindependently selected from halogen, OR⁶, NH₂, NHR⁶, NR⁶R⁶, OH, aromaticring, C(O)R⁶, C₁-C₈ alkyl, C₂-C₈ alkenyl, and C₂-C₈ alkynyl; R⁶ is C₁-C₈alkyl; each R^(T) is independently selected from hydrogen, halogen,Si(R³)₃, C₆H₆, and C₁-C₈ alkyl; u is selected from 0, 1, and 2; v isselected from 0, 1, and 2; w is selected from 0, 1, and 2; s is selectedfrom 0, 1, and 2; t is selected from 0, 1, and 2, and z is selected from0, 1, 2, and 3.

In one aspect, the invention provides a compound of formula I, whereinthe moiety:

In one aspect, the invention provides a compound having a formulaselected from

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof, wherein U is selected from single bond andCR^(2e)R^(2f)—CR^(2g)R^(2h), and R^(2a), R^(2b), R^(2c), R^(2d), t, X,R⁵, R^(2d′), R^(2e), R^(2f), R^(2g), and R^(2h) are the same asdescribed for formula I herein.

In one aspect, the invention provides a compound of formulae Ia, IIa,IIIa, IVa, or Va or a pharmaceutically acceptable salt, hydrate,solvate, or prodrug thereof, wherein the moiety:

In one aspect, the invention provides a compound having the formula Ib:

or a pharmaceutically acceptable salt, hydrate, solvate or prodrugthereof, whereinR^(2a) is selected from hydrogen, OH, NH₂, and C₁-C₈ alkyl;R^(2b) is selected from hydrogen, OH, NH₂, and C₁-C₈ alkyl;R^(2c) is selected from hydrogen, OH, NH₂, and C₁-C₈ alkyl;or taken together two of R^(2a), R^(2b), and R^(2c) form ═O,or taken together two of R^(2a), R^(2b), and R^(2c) form a C₃-C₈cycloalkyl ring,C₄-C₈ cycloalkenyl ring, or 3 to 6 membered saturated or partiallyunsaturated heterocyclic ring, and the remaining R^(2a), R^(2b) orR^(2c) is selected from hydrogen, OH, NH₂, and C₁-C₈ alkyl, or takentogether two of R^(2a), R^(2b), and R^(2c) form an aromatic orheteroaromatic ring and the remaining R^(2a), R^(2b), or R^(2c) isabsent, provided that when two of R^(2a), R^(2b), and R^(2c) form anaromatic ring and the remaining R^(2a), R^(2b), or R^(2c) is absent, tis not 0,wherein said cycloalkyl, cycloalkenyl, heterocyclic, aromatic, andheteroaromatic ring are unsubstituted or substituted with one or moreR^(x);each R^(x) is independently selected from (CH₂)_(z)NH₂, (CH₂)_(z)NHR³,(CH₂)_(z)NR³R³, OR³, OCF₃, OCH₂F, OCHF₂, (CH₂)_(z)-aromatic ring,(CH₂)_(z)-heterocyclic ring, hydroxyl, halogen, C₁-C₈ alkyl, (C₁-C₈alkyl)CF₃, (C₁-C₈ alkyl)OH, C(O)R^(3′)(CH₂)_(z)C(O)NH₂,(CH₂)_(z)C(O)NHR³, (CH₂)_(z)C(O)NR³R³, (CH₂)_(z)NHC(O)R⁴, and(CH₂)_(z)NR⁴C(O)R⁴;or taken together two R^(x) attached to the same carbon atom of acycloalkyl, cycloalkenyl, or heterocyclic ring together form ═O;or taken together two R^(x) form a C₃-C₈ cycloalkyl ring, C₄-C₈cycloalkenyl ring or 3 to 8 membered saturated or partially unsaturatedheterocyclic ring, further wherein said cycloalkyl, cycloalkenyl, andheterocyclic ring are optionally substituted with one or more R^(z);or taken together two R^(x) form an aromatic ring or heteroaromaticring, further wherein said aromatic and heteroaromatic ring areunsubstituted or substituted with one or more R^(z);each R^(z) is independently selected from halogen, C₁-C₄ alkyl, OH, OR³,OCF₃, OCH₂F, OCHF₂, NH₂, NHR³, NR³R³, and C(O)CH₃;R³ is selected from C₁-C₈ alkyl and O(C₁-C₈ alkyl);R⁴ is selected from C₁-C₈ alkyl and CF₃;R⁵ is selected from hydrogen, deuterium, halogen, OH, OR⁶, CF₃, C₁-C₈alkyl, C₂₋₈ alkene, (CH₂)_(s)-aromatic ring, (CH₂)_(s)-heteroaromatic,C₃-C₆ cycloalkyl ring, C₄-C₆ cycloalkenyl ring and 5-6 memberedsaturated, unsaturated, or partially unsaturated heterocyclic ring,wherein said aromatic, heteroaromatic, cycloalkyl, cycloalkenyl, andheterocyclic ring are unsubstituted or substituted with one or moreR^(y) and said alkene is substituted with one or more R^(T);each R^(y) is independently selected from halogen, OR⁶, NH₂, NHR⁶,NR⁶R⁶, OH, C(O)R⁶, C₁-C₈ alkyl, C₂-C₈ alkenyl, and C₂-C₈ alkynyl;R^(T) is independently selected from halogen, hydrogen, C₆H₆, Si(R³)₃and C₁-C₈ alkyl;R⁶ is C₁-C₈ alkyl;s is selected from 0, 1, and 2,t is selected from 0, 1, and 2 andz is selected from 0, 1, 2, and 3.

In one aspect, the invention provides a compound having the formula Ibor a pharmaceutically acceptable salt, hydrate, solvate or prodrugthereof, wherein R^(2a) is selected from hydrogen, OH, NH₂, and C₁-C₈alkyl; R^(2b) is selected from hydrogen, OH, NH₂, and C₁-C₈ alkyl;R^(2c) is selected from hydrogen, OH, NH₂, and C₁-C₈ alkyl;

or taken together two of R^(2a), R^(2b), and R^(2c) form ═O, or takentogether two of R^(2a), R^(2b), and R^(2c) form a C₃-C₈ cycloalkyl ring,C₄-C₈ cycloalkenyl ring, or 3 to 6 membered saturated or partiallyunsaturated heterocyclic ring, and the remaining R^(2a), R^(2b) orR^(2c) is selected from hydrogen, OH, NH₂, and C₁-C₈ alkyl, or takentogether two of R^(2a), R^(2b), and R^(2c) form an aromatic orheteroaromatic ring and the remaining R^(2a), R^(2b), or R^(2c) isabsent,wherein said cycloalkyl, cycloalkenyl, heterocyclic, aromatic, andheteroaromatic ring are unsubstituted or substituted with one or moreR^(x); each R^(x) is independently selected from NH₂, NHR⁶, NR⁶, R⁶,hydroxyl, halogen, C₁-C₈ alkyl, (C₁-C₈)CF₃, (C₁-C₈)OH, C(O)R³, OR³,(CH₂)_(z)C₆H₆, and (CH₂)_(z)NHC(O)R⁴; or taken together two R^(x)attached to the same carbon atom of a cycloalkyl, cycloalkenyl, orheterocyclic ring together form ═O;or taken together two R^(x) form a C₃-C₈ cycloalkyl ring, C₄-C₈cycloalkenyl ring or 3 to 8 membered saturated or partially unsaturatedheterocyclic ring, further wherein said cycloalkyl, cycloalkenyl, andheterocyclic ring are optionally substituted with one or more R^(z);each R^(z) is independently selected from halogen, C₁-C₄ alkyl, OH, NH₂,and C(O)CH₃; R³ is selected from C₁-C₈ alkyl and O(C₁-C₈ alkyl); R⁴ isselected from C₁-C₈ alkyl and CF₃; R⁵ is selected from hydrogen,halogen, OH, OR⁶, CF₃, CH₃, C₂₋₈ alkene, (CH₂)_(s)-aromatic ring,(CH₂)_(s)-heteroaromatic, C₃-C₆ cycloalkyl ring, C₄-C₆ cycloalkenyl ringand 5-6 membered saturated, unsaturated, or partially unsaturatedheterocyclic ring, wherein said aromatic, heteroaromatic, cycloalkyl,cycloalkenyl, and heterocyclic ring are unsubstituted or substitutedwith one or more R^(y) and said alkene is substituted with one or moreR^(T); each R^(y) is independently selected from halogen, OR⁶, NH₂,NHR⁶, NR⁶R⁶, OH, C(O)R⁶, C₁-C₈ alkyl, C₂-C₈ alkenyl, and C₂-C₈ alkynyl;R^(T) is independently selected from halogen, hydrogen, C₆H₆, Si(R³)₃and C₁-C₈ alkyl; R⁶ is C₁-C₈ alkyl; s is selected from 0, 1, and 2, t isselected from 0, 1, and 2 and z is selected from 0, 1, 2, and 3.

In one aspect, the invention provides a compound having the formula Ibb:

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof, wherein R^(2c), R^(2b), R^(2a) and R⁵ are as described herein.

In one aspect, the invention provides a compound having the formulaIbbb:

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof, wherein R^(2c), R^(2b), R^(2a), and R⁵ are as described herein.

In one aspect, the invention provides a compound having the formula IIb:

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof, whereinR^(2a) is selected from hydrogen, halogen, OH, NH₂, and C₁-C₈ alkyl;R^(2b) is selected from hydrogen, halogen, OH, NH₂, and C₁-C₈ alkyl;R^(2c) is selected from hydrogen, halogen, OH, NH₂, and C₁-C₈ alkyl;or alternatively, taken together two of R^(2a), R^(2b), and R^(2c) forma C₃-C₈ cycloalkyl ring, C₄-C₈ cycloalkenyl ring, 3-6 membered saturatedor partially unsaturated heterocyclic ring and the remaining R^(2a),R^(2b), and R^(2c) is selected from hydrogen, halogen, OH, NH₂, andC₁-C₈ alkyl, or taken together two of R^(2a), R^(2b), and R^(2c) form anaromatic or heteroaromatic ring and the remaining R^(2a), R^(2b), orR^(2c) is absent;further, wherein said cycloalkyl, cycloalkenyl, heterocyclic, aromaticand heteroaromatic ring are unsubstituted or substituted with one ormore R^(x);each R^(x) is independently selected from (CH₂)_(z)NH₂, (CH₂)_(z)NHR³,(CH₂)_(z)NR³R³, OR³, OCF₃, OCH₂F, OCHF₂, (CH₂)_(z)-aromatic ring,(CH₂)_(z)-heterocyclic ring, hydroxyl, halogen, C₁-C₈ alkyl, (C₁-C₈alkyl)CF₃, (C₁-C₈ alkyl)OH, C(O)R^(3′)(CH₂)_(z)C(O)NH₂,(CH₂)_(z)C(O)NHR³, (CH₂)_(z)C(O)NR³R³, (CH₂)_(z)NHC(O)R⁴, and(CH₂)_(z)NR⁴C(O)R⁴;R³ is selected from C₁-C₈ alkyl and O(C₁-C₈ alkyl);R⁴ is selected from C₁-C₈ alkyl and CF₃;R⁵ is selected from hydrogen, deuterium, halogen, OH, OR⁶, CF₃, C₁-C₈alkyl, C₂₋₈ alkene, (CH₂)_(s)-aromatic ring, (CH₂)_(s)-heteroaromatic,C₃-C₆ cycloalkyl ring, C₄-C₆ cycloalkenyl ring and 5-6 memberedsaturated, unsaturated, or partially unsaturated heterocyclic ring,wherein said aromatic, heteroaromatic, cycloalkyl, cycloalkenyl, andheterocyclic ring are unsubstituted or substituted with one or moreR^(y) and said alkene is substituted with one or more R^(T);each R^(y) is independently selected from halogen, OR⁶, NH₂, NHR⁶,NR⁶R⁶, OH, aromatic ring, C(O)R⁶, C₁-C₈ alkyl, C₂-C₈ alkenyl, and C₂-C₈alkynyl;R⁶ is C₁-C₈ alkyl;each R^(T) is independently selected from halogen, hydrogen, C₆H₆,Si(R³)₃ and C₁-C₈ alkyl;s is selected from 0, 1, and 2,t is selected from 0, 1, and 2, andz is selected from 0, 1, 2, and 3.

In one aspect, the invention provides a compound having the formula IIbor a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof, wherein R^(2a) is selected from hydrogen, halogen, OH, NH₂, andC₁-C₈ alkyl; R^(2b) is selected from hydrogen, halogen, OH, NH₂, andC₁-C₈ alkyl; R^(2c) is selected from hydrogen, halogen, OH, NH₂, andC₁-C₈ alkyl; or alternatively, taken together two of R^(2a), R^(2b), andR^(2c) form a C₃-C₈ cycloalkyl ring, C₄-C₈ cycloalkenyl ring, 3-6membered saturated or partially unsaturated heterocyclic ring and theremaining R^(2a), R^(2b), and R^(2c) is selected from hydrogen, halogen,OH, NH₂, and C₁-C₈ alkyl, or taken together two of R^(2a), R^(2b), andR^(2c) form an aromatic or heteroaromatic ring and the remaining R^(2a),R^(2b), or R^(2c) is absent;

further, wherein said cycloalkyl, cycloalkenyl, heterocyclic, aromaticand heteroaromatic ring are unsubstituted or substituted with one ormore R^(x); each R^(x) is independently selected from NH₂, NHR³, NR³R³,hydroxyl, halogen, C₁-C₈ alkyl, (C₁-C₈)CF₃, (C₁-C₈)OH, C(O)R³, OR³,(CH₂)_(z)C₆H₆, and (CH₂)_(z)NHC(O)R⁴; R³ is selected from C₁-C₈ alkyland O(C₁-C₈ alkyl); R⁴ is selected from C₁-C₈ alkyl and CF₃; R⁵ isselected from hydrogen, halogen, OH, OR⁶, CF₃, CH₃, C₂₋₈ alkene,(CH₂)_(s)-aromatic ring, (CH₂)_(s)-heteroaromatic, C₃-C₆ cycloalkylring, C₄-C₆ cycloalkenyl ring and 5-6 membered saturated, unsaturated,or partially unsaturated heterocyclic ring, wherein said aromatic,heteroaromatic, cycloalkyl, cycloalkenyl, and heterocyclic ring areunsubstituted or substituted with one or more R^(y) and said alkene issubstituted with one or more R^(T);each R^(y) is independently selected from halogen, OR⁶, NH₂, NHR⁶,NR⁶R⁶, OH, aromatic ring, C(O)R⁶, C₁-C₈ alkyl, C₂-C₈ alkenyl, and C₂-C₈alkynyl;R⁶ is C₁-C₈ alkyl; each R^(T) is independently selected from halogen,hydrogen, C₆H₆, Si(R³)₃ and C₁-C₈ alkyl; s is selected from 0, 1, and 2;t is selected from 0, 1, and 2, andz is selected from 0, 1, 2, and 3.

In one aspect, the invention provides a compound having the formulaIIIb:

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof, whereinR^(2a) is selected from hydrogen, halogen, OH, NH₂, and C₁-C₈ alkyl;R^(2b) is selected from hydrogen, halogen, OH, NH₂, and C₁-C₈ alkyl;R^(2c) is selected from hydrogen, halogen, OH, NH₂, and C₁-C₈ alkyl;R^(2d) is selected from hydrogen, NH₂, and C₁-C₈ alkyl;or alternatively, taken together two of R^(2a), R^(2b), R^(2c) andR^(2d) form a C₃-C₈ cycloalkyl ring, C₄-C₈ cycloalkenyl ring, or 3 to 8membered saturated or partially unsaturated heterocyclic ring and theremaining R^(2a), R^(2b), or R^(2c) is selected from hydrogen, halogen,OH, NH₂, and C₁-C₈ alkyl or R^(2d) is hydrogen, NH₂, or C₁-C₈ alkyl,or taken together two of R^(2a), R^(2b), and R^(2c) form an aromatic orheteroaromatic ring and the remaining R^(2a), R^(2b), or R^(2c) isabsent;further, wherein said cycloalkyl, cycloalkenyl, heterocyclic, aromaticor heteroaromatic ring are unsubstituted or substituted with one or moreR^(x);each R^(x) is independently selected from (CH₂)_(z)NH₂, (CH₂)_(z)NHR³,(CH₂)_(z)NR³R³, OR³, OCF₃, OCH₂F, OCHF₂, (CH₂)_(z)-aromatic ring,(CH₂)_(z)-heterocyclic ring, hydroxyl, halogen, C₁-C₈ alkyl, (C₁-C₈alkyl)CF₃, (C₁-C₈ alkyl)OH, C(O)R^(3′)(CH₂)_(z)C(O)NH₂,(CH₂)_(z)C(O)NHR³, (CH₂)_(z)C(O)NR³R³, (CH₂)_(z)NHC(O)R⁴, and(CH₂)_(z)NR⁴C(O)R⁴;or taken together two R^(x) attached to the same carbon atom of acycloalkyl, cycloalkenyl, or heterocyclic ring together form ═O;or taken together two R^(x) form a C₃-C₈ cycloalkyl ring, C₄-C₈cycloalkenyl ring or 3 to 8 membered saturated or partially unsaturatedheterocyclic ring, further wherein said cycloalkyl, cycloalkenyl, andheterocyclic ring are optionally substituted with one or more R^(z);or taken together two R^(x) form an aromatic ring or heteroaromaticring, further wherein said aromatic and heteroaromatic ring areunsubstituted or substituted with one or more R^(z);each R^(z) is independently selected from halogen, C₁-C₄ alkyl, OH, OR³,OCF₃, OCH₂F, OCHF₂, NH₂, NHR³, NR³R³, and C(O)CH₃;R³ is selected from C₁-C₈ alkyl and O(C₁-C₈ alkyl);R⁴ is selected from C₁-C₈ alkyl and CF₃;R⁵ is selected from hydrogen, deuterium, halogen, OH, OR⁶, CF₃, C₁-C₈alkyl, C₂₋₈ alkene, (CH₂)_(s)-aromatic ring, (CH₂)_(s)-heteroaromatic,C₃-C₆ cycloalkyl ring, C₃-C₆ cycloalkenyl ring and 5-6 memberedsaturated, unsaturated, or partially unsaturated heterocyclic ring,wherein said aromatic, heteroaromatic, cycloalkyl, cycloalkenyl, andheterocyclic ring are unsubstituted or substituted with one or moreR^(y) and said alkene is substituted with one or more R^(T);each R^(y) is independently selected from halogen, OR⁶, NH₂, NHR⁶,NR⁶R⁶, OH, aromatic ring, C(O)R⁶ and C₁-C₈ alkyl;R^(T) is independently selected from halogen, hydrogen, C₆H₆, Si(R³)₃and C₁-C₈ alkyl;R⁶ is C₁-C₈ alkyl;s is selected from 0, 1, and 2,t is selected from 0, 1, and 2, andz is selected from 0, 1, 2, and 3.

In one aspect, the invention provides a compound having the formula IIIbor a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof, wherein R^(2a) is selected from hydrogen, halogen, OH, NH₂, andC₁-C₈ alkyl; R^(2b) is selected from hydrogen, halogen, OH, NH₂, andC₁-C₈ alkyl; R^(2c) is selected from hydrogen, halogen, OH, NH₂, andC₁-C₈ alkyl; R^(2d) is selected from hydrogen, NH₂, and C₁-C₈ alkyl;

or alternatively, taken together two of R^(2a), R^(2b), R^(2c) andR^(2d) form a C₃-C₈ cycloalkyl ring, C₄-C₈ cycloalkenyl ring, or 3 to 8membered saturated or partially unsaturated heterocyclic ring and theremaining R^(2a), R^(2b), or R^(2c) is selected from hydrogen, halogen,OH, NH₂, and C₁-C₈ alkyl or R^(2d) is hydrogen, NH₂, or C₁-C₈ alkyl,or taken together two of R^(2a), R^(2b), and R^(2c) form an aromatic orheteroaromatic ring and the remaining R^(2a), R^(2b), or R^(2c) isabsent; further, wherein said cycloalkyl, cycloalkenyl, heterocyclic,aromatic or heteroaromatic ring are unsubstituted or substituted withone or more R^(x); each R^(x) is independently selected from NH₂, NHR⁶,NR⁶R⁶, hydroxyl, C₁-C₈ alkyl, (C₁-C₈)CF₃, (C₁-C₈)OH, halogen, C(O)R³,OR³, (CH₂)_(z)C₆H₆, and (CH₂)_(z)NHC(O)R⁴; R³ is selected from C₁-C₈alkyl and O(C₁-C₈ alkyl); R⁴ is selected from C₁-C₈ alkyl and CF₃; R⁵ isselected from hydrogen, halogen, OH, OR⁶, CF₃, CH₃, C₂₋₈ alkene,(CH₂)_(s)-aromatic ring, (CH₂)_(s)-heteroaromatic, C₃-C₆ cycloalkylring, C₃-C₆ cycloalkenyl ring and 5-6 membered saturated, unsaturated,or partially unsaturated heterocyclic ring, wherein said aromatic,heteroaromatic, cycloalkyl, cycloalkenyl, and heterocyclic ring areunsubstituted or substituted with one or more R^(y) and said alkene issubstituted with one or more R^(T); each R^(y) is independently selectedfrom halogen, OR⁶, NH₂, NHR⁶, NR⁶R⁶, OH, aromatic ring, C(O)R⁶ and C₁-C₈alkyl; R^(T) is independently selected from halogen, hydrogen, C₆H₆,Si(R³)₃ and C₁-C₈ alkyl; R⁶ is C₁-C₈ alkyl; s is selected from 0, 1, and2; t is selected from 0, 1, and 2, and z is selected from 0, 1, 2, and3.

In one aspect, the invention provides a compound having the formula IVb:

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof, whereinR^(2a) is selected from hydrogen, NH₂, and C₁-C₈ alkyl;R^(2b) is selected from hydrogen, NH₂, and C₁-C₈ alkyl;R^(2c) is selected from hydrogen, NH₂, and C₁-C₈ alkyl;R^(2d) is selected from hydrogen, NH₂, and C₁-C₈ alkyl;R^(2d′) is selected from hydrogen, NH₂, and C₁-C₈ alkyl;or alternatively, taken together two of R^(2a), R^(2b), R^(2c) andR^(2d) form a C₃-C₈ cycloalkyl ring, C₄-C₈ cycloalkenyl ring, 3-6membered saturated, unsaturated, or partially unsaturated heterocyclicring and the remaining R^(2a), R^(2b), or R^(2c) is selected fromhydrogen, halogen, OH, NH₂, and C₁-C₈ alkyl or R^(2d) or R^(2d′) ishydrogen or C₁-C₈ alkyl,or taken together two of R^(2a), R^(2b), and R^(2c) form aromatic orheteroaromatic ring and the remaining R^(2a), R^(2b), and R^(2c) isabsent;further, wherein said cycloalkyl, cycloalkenyl, heterocyclic, aromaticand heteroaromatic ring are unsubstituted or substituted with one ormore R^(x);each R^(x) is independently selected from (CH₂)_(z)NH₂, (CH₂)_(z)NHR³,(CH₂)_(z)NR³R³, OR³, OCF₃, OCH₂F, OCHF₂, (CH₂)_(z)-aromatic ring,(CH₂)_(z)-heterocyclic ring, hydroxyl, halogen, C₁-C₈ alkyl, (C₁-C₈alkyl)CF₃, (C₁-C₈ alkyl)OH, C(O)R^(3′)(CH₂)_(z)C(O)NH₂,(CH₂)_(z)C(O)NHR³, (CH₂)_(z)C(O)NR³R³, (CH₂)_(z)NHC(O)R⁴, and(CH₂)_(z)NR⁴C(O)R⁴;R³ is selected from C₁-C₈ alkyl and O(C₁-C₈ alkyl);R⁴ is selected from C₁-C₈ alkyl and CF₃;R⁵ is selected from hydrogen, deuterium, halogen, OH, OR⁶, CF₃, C₁-C₈alkyl, C₂₋₈ alkene, (CH₂)_(s)-aromatic ring, (CH₂)_(s)-heteroaromatic,C₃-C₆ cycloalkyl ring, C₄-C₆ cycloalkenyl ring, and 5-6 memberedsaturated, unsaturated, or partially unsaturated heterocyclic ring,wherein said aromatic, heteroaromatic, cycloalkyl, cycloalkenyl, andheterocyclic ring are unsubstituted or substituted with one or moreR^(y) and said alkene is substituted with one or more R^(T);each R^(y) is independently selected from halogen, OR⁶, NH₂, NHR⁶,NR⁶R⁶, OH, aromatic ring, C(O)R⁶, C₁-C₈ alkyl, C₂-C₈ alkenyl, and C₂-C₈alkynyl;R⁶ is C₁-C₈ alkyl;each R^(T) is independently selected from halogen, hydrogen, C₆H₆,Si(R³)₃ and C₁-C₈ alkyl;s is selected from 0, 1, and 2,t is selected from 0, 1, and 2, andz is selected from 0, 1, 2, and 3.

In one aspect, the invention provides a compound having the formula IVbor a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof, wherein R^(2a) is selected from hydrogen, NH₂, and C₁-C₈ alkyl;R^(2b) is selected from hydrogen, NH₂, and C₁-C₈ alkyl; R^(2c) isselected from hydrogen, NH₂, and C₁-C₈ alkyl; R^(2d) is selected fromhydrogen, NH₂, and C₁-C₈ alkyl; R^(2d′) is selected from hydrogen, NH₂,and C₁-C₈ alkyl;

or alternatively, taken together two of R^(2a), R^(2b), R^(2c) andR^(2d) form a C₃-C₈ cycloalkyl ring, C₄-C₈ cycloalkenyl ring, 3-6membered saturated, unsaturated, or partially unsaturated heterocyclicring and the remaining R^(2a), R^(2b), or R^(2c) is selected fromhydrogen, halogen, OH, NH₂, and C₁-C₈ alkyl or R^(2d) or R^(2d′) ishydrogen or C₁-C₈ alkyl, or taken together two of R^(2a), R^(2b), andR^(2c) form aromatic or heteroaromatic ring and the remaining R^(2a),R^(2b), and R^(2c) is absent; further, wherein said cycloalkyl,cycloalkenyl, heterocyclic, aromatic and heteroaromatic ring areunsubstituted or substituted with one or more R^(x); each R^(x) isindependently selected from NH₂, NHR³, NR³R³, hydroxyl, halogen, C₁-C₈alkyl, (C₁-C₈)CF₃, (C₁-C₈)OH, C(O)R³, OR³, (CH₂)_(z)C₆H₆, or(CH₂)_(z)NHC(O)R⁴; R³ is selected from C₁-C₈ alkyl and O(C₁-C₈ alkyl);R⁴ is selected from C₁-C₈ alkyl and CF₃; R⁵ is selected from hydrogen,halogen, OH, OR⁶, CF₃, CH₃, C₂₋₈ alkene, (CH₂)_(s)-aromatic ring,(CH₂)_(s)-heteroaromatic, C₃-C₆ cycloalkyl ring, C₄-C₆ cycloalkenylring, and 5-6 membered saturated, unsaturated, or partially unsaturatedheterocyclic ring, wherein said aromatic, heteroaromatic, cycloalkyl,cycloalkenyl, and heterocyclic ring are unsubstituted or substitutedwith one or more R^(y) and said alkene is substituted with one or moreR^(T);each R^(y) is independently selected from halogen, OR⁶, NH₂, NHR⁶,NR⁶R⁶, OH, aromatic ring, C(O)R⁶, C₁-C₈ alkyl, C₂-C₈ alkenyl, and C₂-C₈alkynyl; R⁶ is C₁-C₈ alkyl;each R^(T) is independently selected from halogen, hydrogen, C₆H₆,Si(R³)₃ and C₁-C₈ alkyl; s is selected from 0, 1, and 2; t is selectedfrom 0, 1, and 2, and z is selected from 0, 1, 2, and 3.

In one aspect, the invention provides a compound having the formula Vb:

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof, whereinR^(2a) is selected from hydrogen, NH₂, and C₁-C₈ alkyl;R^(2b) is selected from hydrogen, NH₂, and C₁-C₈ alkyl;R^(2d) is selected from hydrogen, NH₂, and C₁-C₈ alkyl;R⁵ is selected from hydrogen, deuterium, halogen, OH, OR⁶, CF₃, C₁-C₈alkyl, C₂₋₈ alkene, (CH₂)_(s)-aromatic ring, (CH₂)_(s)-heteroaromatic,C₅-C₆ cycloalkyl ring, C₅-C₆ cycloalkenyl ring, and 5-6 memberedsaturated, unsaturated, or partially unsaturated heterocyclic ring,wherein said aromatic, heteroaromatic, cycloalkyl, cycloalkenyl, andheterocyclic ring are unsubstituted or substituted with one or moreR^(y) and said alkene is substituted with one or more R^(T); each R^(y)is independently selected from halogen, OR⁶, NH₂, NHR⁶, NR⁶R⁶, OH,aromatic ring, C(O)R⁶ and C₁-C₈ alkyl;each R^(T) is independently selected from halogen, hydrogen, C₆H₆Si(R³)₃and C₁-C₈ alkyl, and R⁶ is C₁-C₈ alkyl.

In one aspect, the invention provides a compound having the formula (Vb)or a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof, wherein R^(2a) is selected from hydrogen, NH₂, and C₁-C₈ alkyl;R^(2b) is selected from hydrogen, NH₂, and C₁-C₈ alkyl; R^(2d) isselected from hydrogen, NH₂, and C₁-C₈ alkyl; R⁵ is selected fromhydrogen, halogen, OH, OR⁶, CF₃, CH₃, C₂₋₈ alkene, (CH₂)_(s)-aromaticring, (CH₂)_(s)-heteroaromatic, C₅-C₆ cycloalkyl ring, C₅-C₆cycloalkenyl ring, and 5-6 membered heteroaromatic, cycloalkyl,cycloalkenyl, and heterocyclic ring are unsubstituted or substitutedwith one or more R^(y) and said alkene is substituted with one or moreR^(T);

each R^(y) is independently selected from halogen, OR⁶, NH₂, NHR⁶,NR⁶R⁶, OH, aromatic ring, C(O)R⁶ and C₁-C₈ alkyl; each R^(T) isindependently selected from halogen, hydrogen, C₆H₆Si(R³)₃ and R⁶ isC₁-C₈ alkyl.

Furthermore, while all of the compounds of this invention are useful ashistone deacetylase inhibitors, certain classes of compounds, hydrates,solvates, or prodrugs thereof are preferred. The following paragraphsdescribe such preferred classes.

1) R⁵ is selected from hydrogen, deuterium, halogen, OH, OCH₃, CF₃, CH₃,and cyclopropyl;

2) R⁵ is

and m is selected from 1, 2, 3, and 4;

3) R⁵ is

and n is selected from 1, 2, 3, and 4;

4) R⁵ is

each R^(T) is independently selected from halogen, hydrogen, C₆H₆,Si(R³)₃, phenyl; C₁-C₈ alkyl and R₃ is selected from C₁-C₈ alkyl andO(C₁-C₈ alkyl);5) R⁵ is selected from

each Y is independently selected from CH, CR^(y), and N; each j isindependently selected from 0, 1, 2, 3, 4, and 5; each R^(y) isindependently selected from halogen, OR⁶, NHR⁶, NR⁶R⁶, OH, CF₃, aromaticring, C(O)R⁶, C₁-C₈ alkyl, C₂-C₈ alkenyl, and C₂-C₈ alkynyl; and R⁶ isC₁-C₈ alkyl;

6) R⁵ is

each Y is independently selected from CH, CR^(y), and N and providedthat not all Y are N; each R^(y) is independently selected from halogen,OH, OR⁶, NH₂, NHR⁶, NR⁶R⁶, C₁-C₈ alkyl, C₂-C₈ alkenyl, and C₂-C₈alkynyl; each j is independently selected from 0, 1, 2, 3, 4, and 5; andR⁶ is C₁-C₈ alkyl;

7) R⁵ is

each Y is independently selected from CH, CR^(y), NH, NR^(y) and N andprovided that not all Y are N; each R^(y) is independently selected fromhalogen, OH, OR⁶, NH₂, NHR⁶, NR⁶R⁶, C₁-C₈ alkyl, C₂-C₈ alkenyl, andC₂-C₈ alkynyl; j is selected from 0, 1, 2, 3, 4, and 5; and R⁶ is C₁-C₈alkyl;

8) R⁵ is

each Y is independently selected from CH, CR^(y), N, S, and O and notall Y are N; not all Y are O; and not all Y are S; each R^(y) isindependently selected from halogen, OH, OR⁶, NH₂, NHR⁶, NR⁶R⁶, C₁-C₈alkyl, C₂-C₈ alkenyl, and C₂-C₈ alkynyl; j is selected from 0, 1, 2, 3,4, and 5; and R⁶ is C₁-C₈ alkyl;

9) R⁵ is

each Y is independently selected from CH, CR^(y), and N and not all Yare N; each R^(y) is independently selected from halogen, OH, OR⁶, NH₂,NHR⁶, NR⁶R⁶, C₁-C₈ alkyl, C₂-C₈ alkenyl, and C₂-C₈ alkynyl; j isselected from 0, 1, 2, 3, 4, and 5; and R⁶ is C₁-C₈ alkyl;

10) R⁵ is

each Y is independently selected from CH, CR^(y), and S and not all Yare S; each R^(y) is independently selected from halogen, OH, OR⁶, NH₂,NHR⁶, NR⁶R⁶, C₁-C₈ alkyl, C₂-C₈ alkenyl, and C₂-C₈ alkynyl; j isselected from 0, 1, 2, 3, 4, and 5; and R⁶ is C₁-C₈ alkyl;

11) R⁵ is

each Y is independently selected from CH, CR^(y), and O and not all Yare O; each R^(y) is independently selected from halogen, OH, OR⁶, NH₂,NHR⁶, NR⁶R⁶, C₁-C₈ alkyl, C₂-C₈ alkenyl, and C₂-C₅ alkynyl; j isselected from 0, 1, 2, 3, 4, and 5; and R⁶ is C₁-C₈ alkyl;

12) R⁵ is

each Y is independently selected from CH, CR^(y), O and S; and not all Yare O; and not all Y are S; each R^(y) is independently selected fromhalogen, OH, OR⁶, NH₂, NHR⁶, NR⁶R⁶, C₁-C₈ alkyl, C₂-C₈ alkenyl, andC₂-C₈ alkynyl; j is selected from 0, 1, 2, 3, 4, and 5; and R⁶ is C₁-C₈alkyl;

13) R⁵ is

each Y is independently selected from CH, CR^(y), N and S and not all Yare O; each R^(y) is independently selected from halogen, OH, OR⁶, NH₂,NHR⁶, NR⁶R⁶, C₁-C₈ alkyl, C₂-C₈ alkenyl, and C₂-C₈ alkynyl; j isselected from 0, 1, 2, 3, 4, and 5; and R⁶ is C₁-C₈ alkyl;

14) R⁵ is

each Y is independently selected from CH, CR^(y), N and O and not all Yare O; each R^(y) is independently selected from halogen, OH, OR⁶, NH₂,NHR⁶, NR⁶R⁶, C₁-C₈ alkyl, C₂-C₈ alkenyl, and C₂-C₈ alkynyl; j isselected from 0, 1, 2, 3, 4, and 5; and R⁶ is C₁-C₈ alkyl;15) R⁵ is selected from phenyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl,2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 2-pyrazinyl, oxazole,thiazole, and isoxazole;16) R⁵ is selected from phenyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl,2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, and 2-pyrazinyl.17) R⁵ is 4-fluorophenyl;18) R⁵ is 4-pyridinyl;19) R⁵ is 2-thienyl;20) R⁵ is hydrogen;21) R⁵ is selected from oxazole, thiazole, and isoxazole;

22) R⁵ is

each R^(y) is independently selected from halogen, OR⁶, NHR⁶, NR⁶R⁶, OH,CF₃, aromatic ring, C(O)R⁶, C₁-C₈ alkyl, C₂-C₅ alkenyl, and C₂-C₅alkynyl, k is selected from 0, 1, 2, or 3; and R⁶ is C₁-C₈ alkyl;23) R⁵ is selected from hydrogen, deuterium, halogen, OH, OCH₃, CF₃,CH₃, and cyclopropyl;24) R⁵ is selected from OR⁶, wherein R⁶ is C₂-C₈ alkyl, C₂-C₈ alkyl,C₂-C₈ alkene, (CH₂)_(u)-5-6 membered saturated, unsaturated, orpartially unsaturated heterocyclic ring, (CH₂)_(v)—C₄-C₈ cycloalkylring, (C₁-C₈-alkyl)_(w)-C₄-C₈cycloalkenyl ring, (CH₂)_(s)-aromatic ring,and (CH₂)_(s)-heteroaromatic and wherein said heterocyclic, cycloalkyl,cycloalkenyl, heteroaromatic, and aromatic ring are unsubstituted orsubstituted with one or more R^(y) and said alkene is substituted withone or more R^(T);25) R⁵ is selected from hydrogen, deuterium, halogen, OCH₃, CF₃, CH₃ andcyclopropyl;26) R⁶ is CH₃;27) k is 0 or 1;28) k is 1;

29) k is O; 30) R⁵ is

each Z is independently selected from CH₂, CHR^(y), CR^(y)R^(y), O, NH,NR, and S; m is selected from 0, 1, and 2; and each R^(y) isindependently selected from halogen, OR⁶, NHR⁶, NR⁶R⁶, OH, CF₃, aromaticring, C(O)R⁶, C₁-C₈ alkyl, C₂-C₈alkenyl, and C₂-C₈ alkynyl, and k isselected from 0, 1, 2, or 3; and R⁶ is C₁-C₈ alkyl;31) V is C and taken together R^(2a) and R^(2b) are:

o and o′ are each independently selected from 0, 1, and 2; r and r′ areeach independently selected from 0, 1, and 2; q is selected from 0, 1,2, 3, 4, 5, 6, 7, and 8; R^(u) is selected from hydrogen, C₁-C₈ alkyl,(C₁-C₈ alkyl)CF₃, (C₁-C₈ alkyl)OH, and C(O)R^(3a); R^(3a) is C₁-C₈alkyl; v is selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8;R^(2c) is selected from hydrogen, halogen, OH, NH₂, and C₁-C₈ alkyl; andeach R^(x) is independently selected from NH₂, NHR³, NR³R³, OR³,(CH₂)_(z)C₆H₆, hydroxyl, halogen, C₁-C₈ alkyl, (C₁-C₈)CF₃, (C₁-C₈)OH,C(O)R³ and (CH₂)_(z)NHC(O)R⁴or taken together two R^(x) attached to the same carbon atom of acycloalkyl, cycloalkenyl or heterocyclic ring together form ═O; or takentogether two R^(x) form a C₃-C₈ cycloalkyl ring, C₄-C₈ cycloalkenyl ringor 3 to 8 membered saturated or partially unsaturated heterocyclic ring,further wherein said cycloalkyl, cycloalkenyl, and heterocyclic ring areoptionally substituted with one or more R^(z); each R^(z) isindependently selected from halogen, C₁-C₄ alkyl, OH, NH₂, and C(O)CH₃;R³ is selected from C₁-C₈ alkyl and O(C₁-C₈ alkyl) and R⁴ is selectedfrom C₁-C₈ alkyl and CF₃;

32) R⁵ is

each Z is CH₂; and m is selected from 0, 1, and 2;33) R^(2a)R^(2b)R^(2c)C is

o and o′ are each independently selected from 0, 1, and 2; q is selectedfrom 0, 1, 2, 3, 4, 5, 6, 7, and 8; and each R^(x) is independentlyselected from NH₂, NHR³, NR³R³, OR³, (CH₂)_(z)C₆H₆, hydroxyl, halogen,C₁-C₈ alkyl, (C₁-C₈)CF₃, (C₁-C₈)OH, C(O)R³ and (CH₂)_(z)NHC(O)R⁴ ortaken together two R^(x) attached to the same carbon atom of acycloalkyl, cycloalkenyl or heterocyclic ring together form ═O; or takentogether two R^(x) form a C₃-C₈ cycloalkyl ring, C₄-C₈ cycloalkenyl ringor 3 to 8 membered saturated or partially unsaturated heterocyclic ring,further wherein said cycloalkyl, cycloalkenyl, and heterocyclic ring areoptionally substituted with one or more R^(z); each R^(z) isindependently selected from halogen, C₁-C₄ alkyl, OH, NH₂, and C(O)CH₃;R³ is selected from C₁-C₈ alkyl and O(C₁-C₈ alkyl) and R⁴ is selectedfrom C₁-C₈ alkyl and CF₃;34) R^(2a)R^(2b)R^(2c)C is

o and o′ are each independently selected from 0, 1, and 2; q is selectedfrom 0, 1, 2, 3, 4, 5, 6, 7, and 8; and each R^(x) is independentlyselected from NH₂, NHR³, NR³R³, OR³, (CH₂)_(z)C₆H₆, hydroxyl, halogen,C₁-C₈ alkyl, (C₁-C₈)CF₃, (C₁-C₈)OH, C(O)R³ and (CH₂)_(z)NHC(O)R⁴ ortaken together two R^(x) attached to the same carbon atom of acycloalkyl, cycloalkenyl or heterocyclic ring together form ═O; or takentogether two R^(x) form a C₃-C₈ cycloalkyl ring, C₄-C₈ cycloalkenyl ringor 3 to 8 membered saturated or partially unsaturated heterocyclic ring,further wherein said cycloalkyl, cycloalkenyl, and heterocyclic ring areoptionally substituted with one or more R^(z); each R^(z) isindependently selected from halogen, C₁-C₄ alkyl, OH, NH₂, and C(O)CH₃;R³ is selected from C₁-C₈ alkyl and O(C₁-C₈ alkyl) and R⁴ is selectedfrom C₁-C₈ alkyl and CF₃;35) R^(2a)R^(2b)R^(2c)C is

r and r′ are each independently selected from 0, 1, and 2; R^(u) isselected from hydrogen, C₁-C₈ alkyl, (C₁-C₈)CF₃, (C₁-C₈)OH andC(O)R^(3a); R^(3a) is C₁-C₈ alkyl; v is selected from 0, 1, 2, 3, 4, 5,6, 7, and 8; and each R^(x) is independently selected from NH₂, NHR³,NR³R³, OR³, (CH₂)_(z)C₆H₆, hydroxyl, halogen, C₁-C₈ alkyl, (C₁-C₈)CF₃,(C₁-C₈)OH, C(O)R³ and (CH₂)_(z)NHC(O)R⁴ or taken together two R^(x)attached to the same carbon atom of a cycloalkyl, cycloalkenyl orheterocyclic ring together form ═O;or taken together two R^(x) form a C₃-C₈ cycloalkyl ring, C₄-C₈cycloalkenyl ring or 3 to 8 membered saturated or partially unsaturatedheterocyclic ring, further wherein said cycloalkyl, cycloalkenyl, andheterocyclic ring are optionally substituted with one or more R^(z);each R^(z) is independently selected from halogen, C₁-C₄ alkyl, OH, NH₂,and C(O)CH₃; R³ is selected from C₁-C₈ alkyl and O(C₁-C₈ alkyl) and R⁴is selected from C₁-C₈ alkyl and CF₃;36) R^(2a)R^(2b)R^(2c)C is

r and r′ are each independently selected from 0, 1, and 2; R^(u) isselected from hydrogen, C₁-C₈ alkyl, (C₁-C₈)CF₃, (C₁-C₈)OH andC(O)R^(3a); R^(3a) is C₁-C₈ alkyl; v is selected from 0, 1, 2, 3, 4, 5,6, 7, and 8; and each R^(x) is independently selected from NH₂, NHR³,NR³R³, OR³, (CH₂)_(z)C₆H₆, hydroxyl, halogen, C₁-C₈ alkyl, (C₁-C₈)CF₃,(C₁-C₈)OH, C(O)R³ and (CH₂)_(z)NHC(O)R⁴ or taken together two R^(x)attached to the same carbon atom of a cycloalkyl, cycloalkenyl orheterocyclic ring together form ═O;or taken together two R^(x) form a C₃-C₈ cycloalkyl ring, C₄-C₈cycloalkenyl ring or 3 to 8 membered saturated or partially unsaturatedheterocyclic ring, further wherein said cycloalkyl, cycloalkenyl, andheterocyclic ring are optionally substituted with one or more R^(z);each R^(z) is independently selected from halogen, C₁-C₄ alkyl, OH, NH₂,and C(O)CH₃; R³ is selected from C₁-C₈ alkyl and O(C₁-C₈ alkyl) and R⁴is selected from C₁-C₈ alkyl and CF₃;37) R^(2a)R^(2b)R^(2c)C is

and x is selected from 1, 2, 3, and 4;38) R^(2a)R^(2b)R^(2c)C is

w is selected from 1, 2, and 3;

39) U is O and t is 1;

40) R^(2a)R^(2b)R^(2c)V(CH₂)_(t)U is

wherein b is selected from 0, 1, 2, and 3; a is selected from 0, 1, 2,3, 4 5, 6, 7, 8, 9, or 10 and each R^(x) is independently selected fromNH₂, NHR³, NR³R³, OR³, (CH₂)_(z)C₆H₆, hydroxyl, halogen, C₁-C₈ alkyl,(C₁-C₈)CF₃, (C₁-C₈)OH, C(O)R³ and (CH₂)_(z)NHC(O)R⁴; or taken togethertwo R^(x) attached to the same carbon atom of a cycloalkyl, cycloalkenylor heterocyclic ring together form ═O; or taken together two R^(x) forma C₃-C₈ cycloalkyl ring, C₄-C₈ cycloalkenyl ring or 3 to 8 memberedsaturated or partially unsaturated heterocyclic ring, further whereinsaid cycloalkyl, cycloalkenyl, and heterocyclic ring are optionallysubstituted with one or more R^(z); each R^(z) is independently selectedfrom halogen, C₁-C₄ alkyl, OH, NH₂, and C(O)CH₃; R³ is selected fromC₁-C₈ alkyl and O(C₁-C₈ alkyl) and R⁴ is selected from C₁-C₈ alkyl andCF₃;41) R^(2a)R^(2b)R^(2c)V(CH₂)_(t)U is

wherein V is N or CH; T is CH, CR^(z), or N; each R^(z) is independentlyselected from halogen, C₁-C₄ alkyl, OH, OR³, CF₃, OCF₃, OCH₂F, OCHF₂,NH₂, NHR³, NR³R³, and C(O)CH₃; b is 0, 1, 2, 3, or 4; and R³ is selectedfrom C₁-C₈ alkyl and O(C₁-C₈ alkyl).42) R^(2a)R^(2b)R^(2c)V(CH₂)_(t)U is

wherein R^(z) is independently selected from halogen, C₁-C₄ alkyl, OH,OR³, CF₃, OCF₃, OCH₂F, OCHF₂, NH₂, NHR³, NR³R³, and C(O)CH₃; b is 0, 1,2, 3, or 4; and R³ is selected from C₁-C₈ alkyl and O(C₁-C₈ alkyl).43) U is N, t is 1, and R^(2d) is hydrogen, NH₂, or C₁-C₈ alkyl;44) R^(2a)R^(2b)R^(2c)V(CH₂)_(t) is

and each R^(x) is independently selected from NH₂, NHR³, NR³R³, OR³,(CH₂)_(z)C₆H₆, hydroxyl, halogen, C₁-C₈ alkyl, (C₁-C₈)CF₃, (C₁-C₈)OH,C(O)R³ and (CH₂)_(z)NHC(O)R⁴ or taken together two R^(x) attached to thesame carbon atom of a cycloalkyl, cycloalkenyl or heterocyclic ringtogether form ═O or taken together two R^(x) form a C₃-C₈ cycloalkylring, C₄-C₈ cycloalkenyl ring or 3 to 8 membered saturated or partiallyunsaturated heterocyclic ring, further wherein said cycloalkyl,cycloalkenyl, and heterocyclic ring are optionally substituted with oneor more R^(z); each R^(z) is independently selected from halogen, C₁-C₄alkyl, OH, NH₂, and C(O)CH₃; R³ is selected from C₁-C₈ alkyl and O(C₁-C₈alkyl) and R⁴ is selected from C₁-C₈ alkyl and CF₃;45) R^(2a)R^(2b)R^(2c)C(CH₂)_(t) is

46) R^(2a)R^(2b)R^(2c)C(CH₂)_(t) is cyclohexyl;47) U is CH═CH (trans);48) U is CH═CH (cis);49) U is single bond;

50) t is O;

51) t is 1;52) t is 2;53) taken together two of R^(2a), R^(2b), and R^(2c) form ═O and theremaining R^(2a), R^(2b), or R^(2c) is selected from hydrogen, halogen,OH, NH₂, and C₁-C₈ alkyl;54) R^(2a), R^(2b), and R^(2c) are each independently selected fromhydrogen, methyl, and propyl;55) at least two of R^(2a), R^(2b), and R^(2c) are the same;56) R^(2a), R^(2b), and R^(2c) are the same;57) taken together two of R^(2a), R^(2b), and R^(2c) form a saturatedheterocyclic ring selected from tetrahydrofuranyl, tetrahydropyranyl,azetidinyl, piperidinyl, azabicyclo[3.1.0]hexyl,2-oxaspiro[3.3]heptanyl, 2-azaspiro[3.3]heptanyl,8-oxabicyclo[3.2.1]octanyl, and 8-azabicyclo[3.2.1]octanyl;58) taken together two of R^(2a), R^(2b), and R^(2c) form a cycloalkylor cycloalkenyl ring selected from cyclopropyl, cyclobutyl, cyclopentyl,cyclopentenyl, cyclohexyl, and cyclohexenyl;59) taken together two of R^(2a), R^(2b), and R^(2c) form a ring and thering is unsubstituted;60) taken together two of R^(2a), R^(2b), and R^(2c) form a ring and thering is substituted with one or more R^(x), wherein each R^(x) isindependently selected from C(O)R³, (CH₂)_(z)NHC(O)R⁴ and methyl; R³ isselected from C₁-C₈ alkyl and O(C₁-C₈ alkyl) and R⁴ is selected fromC₁-C₈ alkyl and CF₃;61) taken together two of R^(2a), R^(2b), and R^(2c) form a ring and thering is substituted with two R^(x) attached to the same carbon, whichtaken together to form ═O;62) R^(2a), R^(2b), and R^(2c) are each independently selected fromhydrogen, methyl, and fluoro;63) taken together two of R^(2a), R^(2b), and R^(2c) form a cycloalkylring selected from cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl;64) taken together R^(2a) and R^(2b) form a ring and R^(2c) is hydrogen;65) taken together two of R^(2a), R^(2b), and R^(2c) form a ring and thering is substituted with one or more R^(x), wherein each R^(x) isindependently selected from C(O)R³ and methyl;66) R^(2a), R^(2b), R^(2c), and R^(2d) are each independently selectedfrom hydrogen, methyl, and ethyl;67) taken together two of R^(2a), R^(2b), and R^(2c) form a saturatedheterocyclic ring selected from oxetanyl, piperidinyl, piperidinonyl,azetidinyl, pyrrolidinyl, tetrahydropyranyl, azabicyclo[3.1.0]hexanyl,morpholinyl, piperazinyl, 8-oxa-3-azabicyclo[3.2.1]octanyl,hexahydropyridazinyl, 2-oxa-6-azaspiror[3.3]heptanyl,2,6-diazaspiro[3.3]heptanyl, 2-oxaspiro[3.3]heptanyl,2-azaspiroheptanyl, 7-azabicyclo[2.2.1]heptanyl,8-azabicyclo[3.2.1]octanyl, 8-oxabicyclo[3.2.1]octanyl, pyrazolidinyl,and tetrahydrofuranyl;68) taken together two of R^(2a), R^(2b), R^(2c) and R^(2d) form asaturated heterocyclic ring selected from oxetanyl, pyrrolidinyl,azetidinyl, piperidinyl, morpholinyl, piperazinyl,azabicyclo[3.1.0]hexyl, azabicyclo[3.2.1]octanyl, hexahydropyridazinyl,2-oxa-6-azaspiro[3.3]heptanyl, 2,6-diazaspiro[3.3]heptanyl,7-azabicyclo[2.2.1]heptanyl and pyrazolidinyl;69) taken together two of R^(2a), R^(2b), and R^(2c) form a cycloalkylring selected from cyclopropyl, cyclobutyl, and cyclohexyl;70) V is C and taken together two of R^(2a), R^(2b), and R^(2c) form anaromatic ring and the remaining R^(2a), R^(2b), or R^(2c) is absent,wherein said aromatic ring is phenyl, provided that U is not a singlebond and t is 0;71) taken together two of R^(2a), R^(2b), and R^(2c) form a ring and thering is substituted with one or more R^(x), wherein each R^(x) isindependently selected from halogen, NH₂, C(O)R³, (CH₂)_(z)NHC(O)R⁴ andmethyl;72) taken together two of R^(2a), R^(2b), and R^(2c) form a ring and thering is substituted with one or more R^(x), further wherein takentogether two R^(x) form a C₃-C₈ cycloalkyl ring, C₄-C₈ cycloalkenyl ringor 3 to 8 membered saturated or partially unsaturated heterocyclic ring,further wherein said cycloalkyl, cycloalkenyl, and heterocyclic ring areoptionally substituted with one or more R^(z); and each R^(z) isindependently selected from halogen, C₁-C₄ alkyl, OH, NH₂, and C(O)CH₃;73) R^(2a), R^(2b), R^(2c) and R^(2d) are each independently selectedfrom hydrogen and NH₂;74) taken together two of R^(2a), R^(2b), and R^(2c) form a ring and thering is substituted with one or more R^(x), wherein each R^(x) isindependently selected from C(O)R³ and methyl;75) R^(2d) and one of R^(2a) or R^(2b) are selected from hydrogen andNH₂;76) R^(2d) and one of R^(2a) or R^(2b) are both hydrogen;77) R³ or R⁴ is methyl;78) R³ is methyl;79) R³ is ethyl;80) R⁴ is methyl;81) z is 0;82) z is 1;83) R⁵ is selected from hydrogen, thiophenyl, (CH₂)_(s)phenyl,(CH₂)_(s)heteroaryl, pyridinyl, C₂-alkenyl, cyclohexenyl, pyrazinyl, andpyrimidinyl;84) R⁵ is selected from hydrogen, thiophenyl, (CH₂)_(s)phenyl,pyridinyl, C₂-alkenyl, cyclohexenyl, pyrazinyl, and pyrimidinyl;85) R⁵ is selected from 5-pyridinyl, 4-pyridinyl, 3-pyridinyl, and 4-Fphenyl;86) R⁵ is selected from 4-pyridinyl, 3-pyridinyl, and 4-F phenyl;87) R⁵ is 4-pyridinyl;88) R⁵ is 3-pyridinyl;89) R⁵ is 4-F phenyl;90) R⁵ is 5-pyridinyl;91) R⁵ is an unsubstituted ring;92) R⁵ is a ring substituted with one or more R^(y), wherein each R^(y)is independently selected from C(O)R⁶, halogen and methyl;93) R⁵ is selected from hydrogen, thiopheneyl, cyclopentenyl, andphenyl;94) R⁵ is a ring substituted with one or more R^(y), wherein each R^(y)is independently selected from fluoro and chloro;95) R⁵ is selected from hydrogen, thiopheneyl, cyclopropyl, cyclopentylcyclopentenyl, cyclohexenyl, phenyl, pyridinyl, C₂-alkenyl, andpyrazinyl;96) R⁵ is a ring substituted with one or more R^(y), wherein each R^(y)is independently selected from halogen and methyl;97) R⁵ is selected from hydrogen and thiopheneyl;98) R⁵ is unsubstituted thiopheneyl;99) R⁵ is thiopheneyl substituted with one or more R^(y), wherein eachR^(y) is independently selected from C(O)R⁶, halogen or methyl;100) R⁶ is methyl or ethyl;101) s is 0;102) s is 1;103) s is 2;

104) V is C;

105) V is C and taken together R^(2a) and R^(2b) form a saturatedheterocyclic ring:

and R^(2c) is selected from hydrogen, halogen, OH, NH₂, and C₁-C₈ alkyl,further wherein J is selected from N, O, C, and S; when J is N or C,R^(u) is selected from hydrogen, C₁-C₈ alkyl, (C₁-C₈)CF₃, (C₁-C₈)OH,C(O)R^(3a) and when J is O or S, R^(u) is absent; R^(3a) is C₁-C₈ alkyl;R^(x) is selected from NH₂, NHR³, NR³R³, hydroxyl, halogen, C₁-C₈ alkyl,C(O)R³, OR³, (CH₂)_(z)C₆H₆, and (CH₂)_(z)NHC(O)R⁴; R³ is C₁-C₈ alkyl; R⁴is C₁-C₈ alkyl; v is independently selected from 0, 1, 2, 3, 4, 5, 6, 7,and 8, and z is 0, 1, 2, or 3; d and d′ are each independently selectedfrom 0, 1, 2, and 3; or taken together two R^(x) attached to the samecarbon atom of the ring together form ═O; or taken together two R^(x)form a C₃-C₈ cycloalkyl ring, C₄-C₈ cycloalkenyl ring, or 3 to 8membered saturated or partially unsaturated heterocyclic ring, furtherwherein said cycloalkyl, cycloalkenyl, and heterocyclic ring areoptionally substituted with one or more R^(z); and each R^(z) isindependently selected from halogen, C₁-C₄ alkyl, OH, NH₂, and C(O)CH₃;106) V is C and taken together R^(2a) and R^(2b) form a 6-memberedsaturated heterocyclic ring selected from:

and R^(2c) is selected from hydrogen, halogen, OH, NH₂, and C₁-C₈ alkyl,further wherein J is selected from N, O, C, and S; when J is N or C,R^(u) is selected from hydrogen, C₁-C₈ alkyl, (C₁-C₈)CF₃, (C₁-C₈)OH,C(O)R^(3a) and when J is O or S, R^(u) is absent; R^(3a) is C₁-C₈ alkyl;R^(x) is selected from NH₂, NHR³, NR³R³, hydroxyl, halogen, C₁-C₈ alkyl,C(O)R³, OR³, (CH₂)_(z)C₆H₆, and (CH₂)_(z)NHC(O)R⁴; R³ is C₁-C₈ alkyl; R⁴is C₁-C₈ alkyl; v is selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8, and zis 0, 1, 2, or 3; or taken together two R^(x) attached to the samecarbon atom of the ring together form ═O; or taken together two R^(x)form a C₃-C₈ cycloalkyl ring, C₄-C₈ cycloalkenyl ring, or 3 to 8membered saturated or partially unsaturated heterocyclic ring, furtherwherein said cycloalkyl, cycloalkenyl, and heterocyclic ring areoptionally substituted with one or more R^(z); and each R^(z) isindependently selected from halogen, C₁-C₄ alkyl, OH, NH₂, and C(O)CH₃;107) V is C and taken together R^(2a) and R^(2b) form a 5-memberedsaturated heterocyclic ring selected from:

and R^(2c) is selected from hydrogen, halogen, OH, NH₂, and C₁-C₈ alkyl,further wherein J is selected from N, O, C, and S; when J is N or C,R^(u) is selected from hydrogen, C₁-C₈ alkyl, (C₁-C₈)CF₃, (C₁-C₈)OH,C(O)R^(3a) and when J is O or S, R^(u) is absent; R^(3a) is C₁-C₈ alkyl;R^(x) is selected from NH₂, NHR³, NR³R³, hydroxyl, halogen, C₁-C₈ alkyl,C(O)R³, OR³, (CH₂)_(z)C₆H₆, and (CH₂)_(z)NHC(O)R⁴; R³ is C₁-C₈ alkyl; R⁴is C₁-C₈ alkyl; v is selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8, and zis 0, 1, 2, or 3;or taken together two R^(x) attached to the same carbon atom of the ringtogether form ═O;or taken together two R^(x) form a C₃-C₈ cycloalkyl ring, C₄-C₈cycloalkenyl ring, or 3 to 8 membered saturated or partially unsaturatedheterocyclic ring, further wherein said cycloalkyl, cycloalkenyl, andheterocyclic ring are optionally substituted with one or more R^(z); andeach R^(z) is independently selected from halogen, C₁-C₄ alkyl, OH, NH₂,and C(O)CH₃;108) V is C and taken together R^(2a) and R^(2b) form a 4-memberedsaturated heterocyclic ring selected from:

and R^(2c) is selected from hydrogen, halogen, OH, NH₂, and C₁-C₈ alkyl,further wherein J is selected from N, O, C, and S; when J is N or C,R^(u) is selected from hydrogen, C₁-C₈ alkyl, (C₁-C₈)CF₃, (C₁-C₈)OH,C(O)R^(3a) and when J is O or S, R^(u) is absent; R^(3a) is C₁-C₈ alkyl;R^(x) is selected from NH₂, NHR³, NR³R³, hydroxyl, halogen, C₁-C₈ alkyl,C(O)R³, OR³, (CH₂)_(z)C₆H₆, and (CH₂)_(z)NHC(O)R⁴; R³ is C₁-C₈ alkyl; R⁴is C₁-C₈ alkyl; v is selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8, and zis 0, 1, 2, or 3; or taken together two R^(x) attached to the samecarbon atom of the ring together form ═O; or taken together two R^(x)form a C₃-C₈ cycloalkyl ring, C₄-C₈ cycloalkenyl ring, or 3 to 8membered saturated or partially unsaturated heterocyclic ring, furtherwherein said cycloalkyl, cycloalkenyl, and heterocyclic ring areoptionally substituted with one or more R^(z); and each R^(z) isindependently selected from halogen, C₁-C₄ alkyl, OH, NH₂, and C(O)CH₃;109) U is a single bond, t is 0, and V is C;110) V is C and taken together R^(2a) and R^(2b) form a partiallyunsaturated bicyclic ring selected from:

and R^(2c) is selected from hydrogen, halogen, OH, NH₂, and C₁-C₈ alkyl,further wherein J is selected from N, O, C, and S; when J is N or C,R^(u) is selected from hydrogen, C₁-C₈ alkyl, (C₁-C₈)CF₃, (C₁-C₈)OH,C(O)R^(3a) and when J is O or S, R^(u) is absent; R^(3a) is C₁-C₈ alkyl;R^(x) is selected from NH, NHR³, NR³R³, hydroxyl, halogen, C₁-C₈ alkyl,C(O)R³, OR³, (CH₂)_(z)C₆H₆, and (CH₂)_(z)NHC(O)R⁴, R³ is C₁-C₈ alkyl; R⁴is C₁-C₈ alkyl, v is selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8; v′ isselected from 0, 1, 2, 3, and 4; o and o′ are each independentlyselected from 0, 1, 2, and 3; and z is 0, 1, 2, or 3; or taken togethertwo R^(x) attached to the same carbon atom of the ring together form ═O;or taken together two R^(x) form a C₃-C₈ cycloalkyl ring, C₄-C₈cycloalkenyl ring, or 3 to 8 membered saturated or partially unsaturatedheterocyclic ring, further wherein said cycloalkyl, cycloalkenyl, andheterocyclic ring are optionally substituted with one or more R^(z); andeach R^(z) is independently selected from halogen, C₁-C₄ alkyl, OH, NH₂,and C(O)CH₃;111) V is C and taken together R^(2a) and R^(2b) form a saturatedbicyclic ring selected from:

and R^(2c) is selected from hydrogen, halogen, OH, NH₂, and C₁-C₈ alkyl,further wherein J is selected from N, O, C, and S; when J is N or C,R^(u) is selected from hydrogen, C₁-C₈ alkyl, (C₁-C₈)CF₃, (C₁-C₈)OH,C(O)R^(3a) and when J is O or S, R^(u) is absent; R^(3a) is C₁-C₈ alkyl;R^(x) is selected from NH, NHR³, NR³R³, hydroxyl, halogen, C₁-C₈ alkyl,C(O)R³, OR³, (CH₂)_(z)C₆H₆, and (CH₂)_(z)NHC(O)R⁴, R³ is C₁-C₈ alkyl; R⁴is C₁-C₈ alkyl, v is selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8; v′ isselected from 0, 1, 2, 3, and 4; o and o′ are each independentlyselected from 0, 1, 2, and 3; and z is 0, 1, 2, or 3; or taken togethertwo R^(x) attached to the same carbon atom of the ring together form ═O;or taken together two R^(x) form a C₃-C₈ cycloalkyl ring, C₄-C₈cycloalkenyl ring, or 3 to 8 membered saturated or partially unsaturatedheterocyclic ring, further wherein said cycloalkyl, cycloalkenyl, andheterocyclic ring are optionally substituted with one or more R^(z); andeach R^(z) is independently selected from halogen, C₁-C₄ alkyl, OH, NH₂,and C(O)CH₃;112) taken together R^(2a) and R^(2b) form a 10-membered ring systemselected from:

and R^(2c) is selected from hydrogen, halogen, OH, NH₂, and C₁-C₈ alkyl,further wherein J is selected from N, O, C, and S; when J is N or C,R^(u) is selected from hydrogen, C₁-C₈ alkyl, (C₁-C₈)CF₃, (C₁-C₈)OH,C(O)R^(3a) and when J is O or S, R^(u) is absent; R^(3a) is C₁-C₈ alkyl;R^(x) is selected from NH, NHR³, NR³R³, hydroxyl, halogen, C₁-C₈ alkyl,C(O)R³, OR³, (CH₂)_(z)C₆H₆, and (CH₂)_(z)NHC(O)R⁴, R³ is C₁-C₈ alkyl; R⁴is C₁-C₈ alkyl; v is selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8, and zis 0, 1, 2, or 3; or taken together two R^(x) attached to the samecarbon atom of the ring together form ═O; or taken together two R^(x)form a C₃-C₈ cycloalkyl ring, C₄-C₈ cycloalkenyl ring, or 3 to 8membered saturated or partially unsaturated heterocyclic ring, furtherwherein said cycloalkyl, cycloalkenyl, and heterocyclic ring areoptionally substituted with one or more R^(z); and each R^(z) isindependently selected from halogen, C₁-C₄ alkyl, OH, NH₂, and C(O)CH₃;113) taken together two of R^(2a), R^(2b), and R^(2c) form a C₃-C₈cycloalkyl ring, C₄-C₈ cycloalkenyl ring, or a 3 to 8 membered saturatedor partially unsaturated heterocyclic ring, further wherein saidcycloalkyl, cycloalkenyl, and heterocyclic ring are optionallysubstituted with one or more R^(z); and each R^(z) is independentlyselected from halogen, C₁-C₄ alkyl, OH, NH₂, and C(O)CH₃; or takentogether two of R^(2a), R^(2b), and R^(2c) form an aromatic orheteroaromatic ring and the remaining R^(2a), R^(2b), or R^(2c) isabsent, further wherein said aromatic or heteroaromatic ring issubstituted with one or more R^(x);114) V is C and taken together two of R^(2a), R^(2b), and R^(2c) form aring selected from:

and R^(2c) is selected from hydrogen, halogen, OH, NH₂, and C₁-C₈ alkyl,further wherein J is selected from N, O, C, and S; when J is N or C,R^(u) is selected from hydrogen, C₁-C₈ alkyl, (C₁-C₈)CF₃, (C₁-C₈)OH,C(O)R^(3a) and when J is O or S, R^(u) is absent; R^(3a) is C₁-C₈ alkyl;R^(x) is selected from NH, NHR³, NR³R³, hydroxyl, halogen, C₁-C₈ alkyl,C(O)R³, OR³, (CH₂)_(z)C₆H₆, and (CH₂)_(z)NHC(O)R⁴, R³ is C₁-C₈ alkyl; R⁴is C₁-C₈ alkyl; n is selected from 0, 1, 2, and 3; v is selected from 0,1, 2, 3, 4, 5, 6, 7, and 8; and o and o′ are each independently selectedfrom 0, 1, 2, and 3;115) X is selected from hydrogen, deuterium, fluorine, and chlorine;116) X is hydrogen and J is NH₂;117) one X is F and the remaining X are hydrogen;118) the moiety

119) the moiety

120) R^(2a)R^(2b)R^(2c)V(CH₂)_(t)U is

wherein V is N or CH; each R^(z) is independently selected from halogen,C₁-C₄ alkyl, OH, OR³, CF₃, OCF₃, OCH₂F, OCHF₂, NH₂, NHR³, NR³R³, andC(O)CH₃; b is 0, 1, 2, 3, or 4; and R³ is selected from C₁-C₈ alkyl andO(C₁-C₈ alkyl);121) R^(2a)R^(2b)R^(2c)V(CH₂)_(t)U is

wherein V is N or CH; each R^(z) is independently selected from halogen,C₁-C₄ alkyl, OH, OR³, CF₃, OCF₃, OCH₂F, OCHF₂, NH₂, NHR³, NR³R³, andC(O)CH₃; b is 0, 1, 2, 3, or 4; and R³ is selected from C₁-C₈ alkyl andO(C₁-C₈ alkyl);122) R^(2a)R^(2b)R^(2c)V(CH₂)_(t)U is

wherein V is N or CH; each R^(z) is independently selected from halogen,C₁-C₄ alkyl, OH, OR³, CF₃, OCF₃, OCH₂F, OCHF₂, NH₂, NHR³, NR³R³, andC(O)CH₃; b is 0, 1, 2, 3, or 4; and R³ is selected from C₁-C₈ alkyl andO(C₁-C₈ alkyl);

123) V is N;

124) the moiety:

125) any compound exemplified;126) the compound is a pharmaceutically acceptable salt;127) the compound is a hydrate;128) the compound is a solvate; and129) the compound is a prodrug.

It will be understood that the above classes may be combined to formadditional classes, as for example the combination of selections of twoor more substituents forming additional classes. Illustrative examplesof combinations of the classes above forming additional classes are:

200) the combination of any of classes 31), or 33)-38) with classes 39),43), and 109);201) the combination of any of classes 31), or 33)-38) with classes47)-49);202) the combination of the combination of 201) with classes 50)-52);203) the combination of any of classes 40)-42), 70), or 120)-122) withany of classes 118) or 119);204) the combination of any of classes 53)-69), 71)-72) or 112)-113)with any of classes 39), 43), and 109);205) the combination of the combination of 204) with any of classes 123)or 104);206) the combination of any of classes 53)-69), 71)-72), or 112)-113)with any of classes 47)-49);207) the combination of the combination of 206) with classes 50)-52);208) the combination of the combination of 207) with any of classes 123)or 104);209) the combination of any of classes of 105)-108), 110)-111), or 114)with any of classes 39), 43), and 109);210) the combination of any of classes of 105)-108), 110)-111), or 114)with any of classes 47)-49);211) the combination of the combination of 210) with any of classes50)-52);212) the combination of any of classes 73)-76) and class 123);213) the combination of the combination of 212) with any of classes50)-52);214) the combination of any of classes 44)-46) with any of classes47)-49);215) the combination of any of the combinations of 200), 202), 203),205), 208), 209), 211), 213), or 214) with any of classes 118)-119) and124);216) the combination of any of the combinations of 200), 202), 203),205), 208), 209), 211), 213), or 214) with class 116);217) the combination of any of classes 40)-42), 70), or 120)-122) withclass 116)218) the combination of any of the combinations of 203) or 215)-217)with 1)-25), 30), 32), and 83)-99).

The above classes and combinations apply to formulae I, Ia, IIa, IIIa,IVa, Va, Ib, Ibb, Ibbb, IIb, IIIb, IVb, and Vb. It is also noted thatcompounds of formulae Ia, IIa, IIIa, IVa, Va, Ib, Ibb, Ibbb, IIb, IIIb,IVb, and Vb are subsets of compounds of formula I. Features describedherein for compounds of formula I apply equally to compounds of formulaeIa, IIa, IIIa, IVa, Va, Ib, Ibb, Ibbb, IIb, IIIb, IVb, and Vb.

In one aspect of the invention, for formula I, Ia and Ib, the compoundis not N-(2-amino-5-(thiophen-2-yl)phenyl)acetamide. In one aspect ofthe invention, for a compound of formulae I or Ia, when R⁵ isthiopheneyl and X is H, then U—(CH₂)_(t)—VR^(2a)R^(2b)R^(2c) is not CH₃.In one aspect of the invention, for a compound of formulae I or Ia,U—(CH₂)_(t)—VR^(2a)R^(2b)R^(2c) is not CH₃. In one aspect of theinvention, for formula Ib, when R⁵ is thiopheneyl, thenR^(2a)R^(2b)R^(2c)(CH₂)_(t) is not CH₃. In one aspect of the invention,for formula Ib, R^(2a)R^(2b)R^(2c)(CH₂)_(t) is not CH₃.

In one aspect of the invention, for a compound of formula I or Ia, whenU is a single bond, t is 0, V is C, then two of R^(2a), R^(2b), andR^(2c) do not form a partially unsaturated heterocylic ring. In oneaspect of the invention, for a compound of formula I or Ia, when U issingle bond, t is 0, V is C, then two of R^(2a), R^(2b), and R^(2c) donot form a pyridinone, 2,3,4,9-tetrahydrocarbazole, benzoimidazole, or1,2,3,4-tetrahydroquinoline ring. In one aspect of the invention, for acompound of formula Ib, when t is 0 then two of R^(2a), R^(2b), andR^(2c) do not form a partially unsaturated heterocylic ring. In oneaspect, of the invention for a compound of formula Ib, when t is 0, thentwo of R^(2a), R^(2b), and R^(2c) do not form a pyridinone,2,3,4,9-tetrahydrocarbazole, benzoimidazole, or1,2,3,4-tetrahydroquinoline ring.

In one aspect of the invention, for a compound of formula I or Ia, whenU is a single bond, t is 0, V is C, and two of R^(2a), R^(2b), andR^(2c) form a piperidine ring, then R^(x) is not benzyl, benzoyl, orbenzodioxole. In one aspect of the invention, for a compound of formulaIb, when t is 0 and two of R^(2a), R^(2b), and R^(2c) form a piperidinering, then R^(x) is not benzyl, benzoyl, or benzodioxole.

In one aspect of the invention, for a compound of formula I or Ia, whenU is a single bond, t is 0, V is N, and two of R^(2a), R^(2b), andR^(2c) form a piperazine ring, then R^(x) is not benzyl or benzodioxoleand when U is NR^(2d) and taken together R^(2d) with one of R^(2a),R^(2b), and R^(2c) form a piperazine ring, then R^(x) is not benzyl orbenzodioxole. In one aspect of the invention for a compound of formulaIIIa or IIIb, when one of R^(2a), R^(2b), and R^(2c) taken together withR^(2d) forms a piperazine ring, then R^(x) is not benzyl orbenzodioxole.

In one aspect, the invention does not include a compound having themoiety:

wherein the phenyl ring is unsubstituted or substituted.

In one aspect of the invention, the compound is not1-methylpiperidin-2-yl)methyl(2-amino-5-(thiophen-2-yl)phenyl)carbamate,1-(2-amino-5-(thiophen-2-yl)phenyl)-3-phenylurea,1-(2-amino-5-(thiophen-2-yl)phenyl)-3-cyclohexylurea,1-(2-amino-5-(thiophen-2-yl)phenyl)-3-(pyrrolidin-3-yl)urea, phenyl(2-amino-5-(thiophen-2-yl)phenyl)carbamate,N-(2-amino-5-(pyrimidin-5-yl)phenyl)cyclohexanecarboxamide,N-(2-amino-5-phenethylphenyl)cyclohexanecarboxamide, andN-(2-amino-5-(thiophen-2-yl)phenyl)-2-propylpentanamide.

In addition to those compounds presented in the examples, the followingcompounds further illustrate the scope of the present invention:

TABLE 1 Cmpd No. structure 1

2

3

4

5

6

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

72

73

74

75

76

77

78

79

80

81

82

83

85

86

87

88

89

90

91

92

93

94

95

96

97

98

100

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180

181

182

183

185

186

187

188

189

190

191

192

193

194

200

195

196

197

198

199

201

202

203

204

205

206

207

208

209

210

211

212

213

214

215

216

217

218

219

220

221

222

223

224

225

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

247

In one aspect, the subject to be administered compounds of thisinvention is human.

Compounds of the invention can be prepared according to methods known inthe art. The schemes below depict general routes for the preparation ofcompounds of the invention.

More specifically, compounds of the formula (A) can be preparedaccording to procedures similar to those depicted in retro-synthesicSchemes 1A-3A.

In Scheme 1A, Step 1 is the aniline Boc protection of compound 1x toform compound 2a using sodium hydride and di-tert-butyl dicarbonate.Step 2(a or b) is a Suzuki reaction of compound 2(a or b) with theappropriate boronic acid, or pinacol boronate or bromo coupling partnerto form compound 3x. Compound 3x is then reduced under hydrogenatmosphere using palladium on carbon in Step 3. Alternatively, thereduction can also be carried out using ferric chloride and hydrazinehydrate. Step 4 is the amide coupling of compound 4x with a carboxylicacid using HATU in the presence of Hünigs base. Alternatively, compound4x can be coupled with an acyl chloride in the presence oftriethylamine. Finally, Step 5 is the removal of the Boc protectinggroup in the presence of trifluoroacetic acid or hydrochloric acid toafford a compound of the invention e.g., compound (45).

In Scheme 2A, Step 1 is a Suzuki reaction of compound 1y with a boronicacid or pinacol borate group to form compound 2y. In step 2, compound 2yis then coupled to an alcohol using triphosgene to form compound 3y.Alternatively, the coupling can be done using a chloroformate in thepresence of triethylamine. Step 3 is the removal of the Boc protectinggroup to afford compound 4y. Compound 4y can then be reduced underhydrogen atmosphere using palladium on carbon to yield compound (75) inStep 4. Alternatively, the nitro group reduction can be carried outusing zinc and ammonium formate. Compound 4y can also be reductivelyaminated using aqueous formaldehyde and sodium cyanoborohydride toafford compound 6y. Compound 4y can also be acetylated using aceticanhydride in the presence of triethylamine to afford compound 5y. Thenitro group is reduced to afford a compound of the invention e.g.,compound (73) in step 7 and e.g., compound (74) in step 8.

In Scheme 3A, in step 1, compound 1z is coupled to an amine usingtriphosgene to form compound 2z. Alternatively, the coupling can be doneusing a readily available isocyanate or a carbamic chloride in thepresence of triethylamine. Step 2 is the removal of the Boc protectinggroup to afford compound 3z. Compound 3z can then be reduced underhydrogen atmosphere using palladium on carbon to yield compound 4z inStep 3. Alternatively, the nitro group reduction can be carried outusing zinc and ammonium formate. Compound 3z can also be reductivelyaminated using aqueous formaldehyde and sodium cyanoborohydride toafford compound 6z. Compound 3z can also be acetylated using aceticanhydride in the presence of triethylamine to afford compound 5z. Thenitro group is in turn reduced to afford a compound of the inventione.g., compound (156) in step 6 and compound (155) in step 7.

Selected Methods of the Invention

Compounds of the invention are inhibitors of class I histonedeacetylases (HDAC) and are useful for promoting cognitive function andenhancing learning and memory formation. As a result, these compoundsare useful in treating, alleviating, and/or preventing variousconditions, including e.g., neurological disorders, memory and cognitivefunction disorders/impairments, extinction learning disorders, fungaldiseases, inflammatory diseases, hematological diseases, and neoplasticdiseases in humans and animals.

Inhibition of Histone Deacetylase

The compounds of the present invention are useful in a variety ofapplications for human and animal health. The compounds of the inventionare histone deacetylase (HDAC) inhibitors. A histone deacetylaseinhibitor as used herein is a compound that inhibits, reduces, orotherwise modulates the activity of histone deacetylase. HDACs catalyzethe removal of acetyl groups from lysine residues on proteins, includinghistones. HDAC inhibitors also show diverse biological functionsincluding effecting gene expression, cell differentiation, cell cycleprogression, growth arrest, and/or apoptosis. (J. Med. Chem. 2003,46:5097 and Curr. Med. Chem. 2003, 10:2343). In various embodiments, thecompounds of the invention reduce HDAC activity by at least about 50%,at least about 75%, or at least about 90% or more. In furtherembodiments, HDAC activity is reduced by at least about 95% or at leastabout 99% or more.

One aspect of the invention provides a method of inhibiting histonedeacetylase in a cell, comprising contacting a cell in which inhibitionof histone deacetylase is desired with an inhibition effective amount ofa compound of the invention or a composition thereof. Because compoundsof the invention inhibit histone deacetylase(s), they are usefulresearch tools for in vitro study of the role of histone deacetylase inbiological processes. Accordingly, in one aspect of the invention, thestep of contacting the cell is performed in vitro.

The term an “inhibiting effective amount” is meant to denote a dosagesufficient to cause inhibition of activity of one or more histonedeacetylase in a cell, which cell can be in a multicellular organism.The multicellular organism can be a plant, a fungus, or an animal,preferably a mammal, more preferably a human. The fungus may beinfecting a plant or a mammal, preferably a human, and could thereforebe located in and/or on the plant or mammal. If the histone deacetylaseis in a multicellular organism, the method according to this aspect ofthe invention comprises administering to the organism a compound orcomposition of the invention. Measurement of the effect of a compound ofthe invention on the enzymatic activity of a histone deacetylase isachieved using known methodologies. For example, Bradner, J. et al.Nature Chemical Biology, Vol. 6, March 2010, 238-243.

The potential of HDAC inhibitors is tremendous, but the development ofclinical compounds will likely require the design of isoform selectivecompounds to minimize side effect issues e.g., fatigue, anorexia,hematological and GI-toxicity. Isoform specific HDAC inhibitors provideadvantages by reducing toxicities associated with inhibition of otherHDACs. Specific HDAC inhibitors provide a higher therapeutic index,resulting in better tolerance by patients during chronic or long termtreatment. While several HDAC inhibitors are now in the clinic, most ofthese do not show significant selectivity for individual HDAC isoforms.

HDACs are classified into four classes depending on sequence identity,domain, organization, and function. Compounds of the invention arepredominately inhibitors of class I histone deacetylases. Class Ienzymes (HDACs 1, 2, 3, and 8) range in size from 42-55 kDa, and arehomologs of yeast Rpd3. They are ubiquitously expressed, predominantlynuclear and mainly function as transcriptional corepressors.

In some other embodiments, the compound reduces the activity of fewerthan all histone deacetylases in the cell. In certain embodiments, thecompound reduces the activity of one histone deacetylase (e.g., HDAC1)or a sub-group of histone deacetylases (e.g., HDAC1, HDAC2, and HDAC3)to a greater extent than other histone deacetylases. Where the compoundpreferentially reduces the activity of a sub-group of histonedeacetylases, the reduction in activity of each member of the sub-groupmay be the same or different.

In certain embodiments, the present invention relates to theaforementioned compound, wherein the compounds of the invention areselective HDAC class 1 inhibitors. In one aspect, a compound is a HDAC2inhibitor. The compound may be a selective HDAC2 inhibitor. In otherembodiments, the compound is a non-selective inhibitor of HDAC2. Inanother aspect, the compound is a HDAC1 inhibitor. The compound may be aselective HDAC1 inhibitor. In other embodiments, the compound is anon-selective inhibitor of HDAC1. In one aspect, a compound is a HDAC3inhibitor. The compound may be a selective HDAC3 inhibitor. In otherembodiments, the compound is a non-selective inhibitor of HDAC3.

In yet another embodiment, a compound is a HDAC1/HDAC2 selectiveinhibitor. In another embodiment, the compound is a HDAC1/HDAC2/HDAC3selective inhibitor.

In one embodiment, a compound selective for HDAC1 will have at leastabout 2-fold (e.g., at least about 5-fold, 10-fold, 15-fold, or 20-fold)greater activity to inhibit HDAC1 as compared to one or more other HDACs(e.g., one or more HDACs of class I or II). In one embodiment, acompound selective for HDAC2 will have at least about 2-fold (e.g., atleast about 5-fold, 10-fold, 15-fold, or 20-fold) greater activity toinhibit HDAC2 as compared to one or more other HDACs (e.g., one or moreHDACs of class I or II). In one embodiment, a compound selective forHDAC3 will have at least about 2-fold (e.g., at least about 5-fold,10-fold, 15-fold, or 20-fold) greater activity to inhibit HDAC3 ascompared to one or more other HDACs (e.g., one or more HDACs of class Ior II).

In one embodiment, a compound selectively inhibits at least one class IHDAC enzyme with an IC₅₀ value greater than 0.0000001 μM and less thanor equal to 0.1 μM, 1 μM, 5 μM, or 30 μM. In another embodiment, acompound selectively inhibits HDAC1 with an IC₅₀ value greater than0.0000001 μM and less than or equal to 0.1 μM, 1 μM, 5 μM, or 30 μM. Inanother embodiment, a compound selectively inhibits HDAC2 with an IC₅₀value greater than 0.0000001 μM and less than or equal to 0.1 μM, 1 μM,5 μM, or 30 μM. In another embodiment, a compound selectively inhibitsHDAC3 with an IC₅₀ value greater than 0.0000001 μM and less than orequal to 0.1 μM, 1 μM, 5 μM, or 30 μM. In one embodiment, a compoundselectively inhibits at least two class I HDAC enzymes with IC₅₀ valuesgreater than 0.0000001 μM and less than or equal to 0.1 μM, 1 μM, 5 μM,or 30 μM. In one embodiment, a compound selectively inhibits at leastthree class I HDAC enzymes with IC₅₀ values greater than 0.0000001 μMand less than or equal to 0.1 μM, 1 μM, 5 μM, or 30 μM.

Neurological Disorders

In one aspect, the invention provides methods and compositions fortreating, alleviating, and/or preventing neurological disorders.

Recent reports have detailed the importance of histone acetylation incentral nervous system (“CNS’) functions such as neuronaldifferentiation, memory formation, drug addiction, and depression(Citrome, Psychopharmacol. Bull. 2003, 37, Suppl. 2, 74-88; Johannessen,CNS Drug Rev. 2003, 9, 199-216; Tsankova et al., 2006, Nat. Neurosci. 9,519-525).

In one aspect, the invention provides methods and compositions fortreating, alleviating, and/or preventing neurological disorders. Theterm “neurological disorder” as used herein includes neurologicaldiseases, neurodegenerative diseases and neuropsychiatric disorders. Aneurological disorder is a condition having as a component a central orperipheral nervous system malfunction. Neurological disorders may causea disturbance in the structure or function of the nervous systemresulting from developmental abnormalities, disease, genetic defects,injury or toxin. These disorders may affect the central nervous system(e.g., the brain, brainstem and cerebellum), the peripheral nervoussystem (e.g., the cranial nerves, spinal nerves, and sympathetic andparasympathetic nervous systems) and/or the autonomic nervous system(e.g., the part of the nervous system that regulates involuntary actionand that is divided into the sympathetic and parasympathetic nervoussystems).

As used herein, the term “neurodegenerative disease” implies anydisorder that might be reversed, deterred, managed, treated, improved,or eliminated with agents that stimulate the generation of new neurons.Examples of neurodegenerative disorders include: (i) chronicneurodegenerative diseases such as familial and sporadic amyotrophiclateral sclerosis (FALS and ALS, respectively), familial and sporadicParkinson's disease, Huntington's disease, familial and sporadicAlzheimer's disease, multiple sclerosis, muscular dystrophy,olivopontocerebellar atrophy, multiple system atrophy, Wilson's disease,progressive supranuclear palsy, diffuse Lewy body disease,corticodentatonigral degeneration, progressive familial myoclonicepilepsy, strionigral degeneration, torsion dystonia, familial tremor,Down's Syndrome, Gilles de la Tourette syndrome, Hallervorden-Spatzdisease, diabetic peripheral neuropathy, dementia pugilistica, AIDSDementia, age related dementia, age associated memory impairment, andamyloidosis-related neurodegenerative diseases such as those caused bythe prion protein (PrP) which is associated with transmissiblespongiform encephalopathy (Creutzfeldt-Jakob disease,Gerstmann-Straussler-Scheinker syndrome, scrapic, and kuru), and thosecaused by excess cystatin C accumulation (hereditary cystatin Cangiopathy); and (ii) acute neurodegenerative disorders such astraumatic brain injury (e.g., surgery-related brain injury), cerebraledema, peripheral nerve damage, spinal cord injury, Leigh's disease,Guillain-Barre syndrome, lysosomal storage disorders such aslipofuscinosis, Alper's disease, restless leg syndrome, vertigo asresult of CNS degeneration; pathologies arising with chronic alcohol ordrug abuse including, for example, the degeneration of neurons in locuscoeruleus and cerebellum, drug-induced movement disorders; pathologiesarising with aging including degeneration of cerebellar neurons andcortical neurons leading to cognitive and motor impairments; andpathologies arising with chronic amphetamine abuse to includingdegeneration of basal ganglia neurons leading to motor impairments;pathological changes resulting from focal trauma such as stroke, focalischemia, vascular insufficiency, hypoxic-ischemic encephalopathy,hyperglycemia, hypoglycemia or direct trauma; pathologies arising as anegative side-effect of therapeutic drugs and treatments (e.g.,degeneration of cingulate and entorhinal cortex neurons in response toanticonvulsant doses of antagonists of the NMDA class of glutamatereceptor) and Wernicke-Korsakoff's related dementia. Neurodegenerativediseases affecting sensory neurons include Friedreich's ataxia,diabetes, peripheral neuropathy, and retinal neuronal degeneration.Other neurodegenerative diseases include nerve injury or traumaassociated with spinal cord injury.

Neurodegenerative diseases of limbic and cortical systems includecerebral amyloidosis, Pick's atrophy, and Retts syndrome. The foregoingexamples are not meant to be comprehensive but serve merely as anillustration of the term “neurodegenerative disorder.”

In some instances the neurological disorder is a neuropsychiatricdisorder, which refers to conditions or disorders that relate to thefunctioning of the brain and the cognitive processes or behavior.Neuropsychiatric disorders may be further classified based on the typeof neurological disturbance affecting the mental faculties. The term“neuropsychiatric disorder,” considered here as a subset of“neurological disorders,” refers to a disorder which may be generallycharacterized by one or more breakdowns in the adaptation process. Suchdisorders are therefore expressed primarily in abnormalities of thought,feeling and/or behavior producing either distress or impairment offunction (i.e., impairment of mental function such with dementia orsenility). Currently, individuals may be evaluated for variousneuropsychiatric disorders using criteria set forth in the most recentversion of the American Psychiatric Association's Diagnostic andStatistical Manual of Mental Health (DSM-IV).

One group of neuropsychiatric disorders includes disorders of thinkingand cognition, such as schizophrenia and delirium. A second group ofneuropsychiatric disorders includes disorders of mood, such as affectivedisorders and anxiety. A third group of neuropsychiatric disordersincludes disorders of social behavior, such as character defects andpersonality disorders. A fourth group of neuropsychiatric disordersincludes disorders of learning, memory, and intelligence, such as mentalretardation and dementia. Accordingly, neuropsychiatric disordersencompass schizophrenia, delirium, attention deficit disorder (ADD),schizoaffective disorder, Alzheimer's disease, Rubinstein-Taybisyndrome, depression, mania, attention deficit disorders, drugaddiction, dementia, agitation, apathy, anxiety, psychoses, personalitydisorders, bipolar disorders, unipolar affective disorder,obsessive-compulsive disorders, eating disorders, post-traumatic stressdisorders, irritability, adolescent conduct disorder and disinhibition.

In one embodiment, the neurological disorder is Alzheimer's disease,Huntington's disease, seizure-induced memory loss, schizophrenia,Rubinstein Taybi syndrome, Rett Syndrome, Fragile X, Lewy body dementia,vascular dementia, ADHD, ADD, dyslexia, bipolar disorder and social,cognitive and learning disorders associated with autism, traumatic headinjury, or attention deficit disorder.

In another embodiment, the neurological disorder is an anxiety disorder,conditioned fear response, panic disorder, obsessive compulsivedisorder, post-traumatic stress disorder, phobia, social anxietydisorder, or substance dependence recovery.

In some embodiments neurological disorders are treated or prevented bydecreasing the amount of DNA damage within the neuronal cell. In someembodiments neurological disorders are treated or prevented byincreasing histone deacetylase activity within the neuronal cell. Insome embodiments neurological disorders are treated or prevented bydecreasing histone acetyl transferase activity within the neuronal cell.In some embodiments neurological disorders are treated or prevented byincreasing the activity of class I histone deacetylases.

Enhancing Cognitive Function

In one aspect, the invention provides methods and compositions forpromoting cognitive function and enhancing learning and memory formationin both normal subjects as well as those suffering from memory loss andcognitive function disorders/impairments. A normal subject, as usedherein, is a subject that has not been diagnosed with a disorderassociated with impaired cognitive function. “Cognitive function” refersto mental processes of a subject relating to information gatheringand/or processing; the understanding, reasoning, and/or application ofinformation and/or ideas; the abstraction or specification of ideasand/or information; acts of creativity, problem-solving, and possiblyintuition; and mental processes such as learning, perception, and/orawareness of ideas and/or information. The mental processes are distinctfrom those of beliefs, desires, and the like.

Memory Disorders/Impairment

Transcription is thought to be a key step for long-term memory processes(Alberini, 2009, Physiol. Rev. 89, 121-145). Transcription is promotedby specific chromatin modifications, such as histone acetylation, whichmodulate histone-DNA interactions (Kouzarides, 2007, Cell, 128:693-705).Modifying enzymes, such as histone acetyltransferases (HATs) and histonedeacetylases (HDACs), regulate the state of acetylation on histonetails. In general, histone acetylation promotes gene expression, whereashistone deacetylation leads to gene silencing. Numerous studies haveshown that a potent HAT, cAMP response element-binding protein(CREB)-binding protein (CBP), is necessary for long-lasting forms ofsynaptic plasticity and long term memory (for review, see Barrett, 2008,Learn Mem 15:460-467).

In contrast, HDACs have been shown to be powerful negative regulators oflong-term memory processes. Nonspecific HDAC inhibitors enhance synapticplasticity as well as long-term memory (Levenson et al., 2004, J. Biol.Chem. 279:40545-40559; Lattal et al., 2007, Behav Neurosci121:1125-1131; Vecsey et al., 2007, J. Neurosci 27:6128; Bredy, 2008,Learn Mem 15:460-467; Guan et al., 2009, Nature 459:55-60; Malvaez etal., 2010, Biol. Psychiatry 67:36-43; Roozendaal et al., 2010, J.Neurosci. 30:5037-5046). For example, HDAC inhibition can transform alearning event that does not lead to long-term memory into a learningevent that does result in significant long-term memory (Stefanko et al.,2009, Proc. Natl. Acad. Sci. USA 106:9447-9452). Furthermore, HDACinhibition can also generate a form of long-term memory that persistsbeyond the point at which normal memory fails. HDAC inhibitors have beenshown to ameliorate cognitive deficits in genetic models of Alzheimer'sdisease (Fischer et al., 2007, Nature 447:178-182; Kilgore et al., 2010,Neuropsychopharmacology 35:870-880). These demonstrations suggest thatmodulating memory via HDAC inhibition have considerable therapeuticpotential for many memory and cognitive disorders.

Currently, the role of individual HDACs in long-term memory has beenexplored in two recent studies. Kilgore et al. 2010,Neuropsychopharmacology 35:870-880 revealed that nonspecific HDACinhibitors, such as sodium butyrate, inhibit class I HDACs (HDAC1,HDAC2, HDAC3, HDAC8) with little effect on the class IIa HDAC familymembers (HDAC4, HDAC5, HDAC7, HDAC9). This suggests that inhibition ofclass I HDACs may be critical for the enhancement of cognition observedin many studies. Indeed, forebrain and neuron specific over expressionof HDAC2, but not HDAC1, decreased dendritic spine density, synapticdensity, synaptic plasticity and memory formation. (Guan et al., 2009,Nature, 459:55-60). In contrast, HDAC2 knockout mice exhibited increasedsynaptic density, increased synaptic plasticity and increased dendriticdensity in neurons. These HDAC2 deficient mice also exhibited enhancedlearning and memory in a battery of learning behavioral paradigms. Thiswork demonstrates that HDAC2 is a key regulator of synaptogenesis andsynaptic plasticity. Additionally, Guan et al. showed that chronictreatment of mice with SAHA (an HDAC1, 2, 3, 6, 8 inhibitor) reproducedthe effects seen in the HDAC2 deficient mice and recused the cognitiveimpairment in the HDAC2 overexpression mice. The inhibition of the HDAC2(selectively or in combination with other class I HDACs) is anattractive target for enhancing cognition and facilitating the learningprocess through increasing synaptic and dendritic density in neuronalcell populations.

HDAC3 is the most highly expressed class I HDAC throughout the brain,including the hippocampus (Broide et al., 2007, J. Mol. Neurosci.31:47-58). HDAC3 alters gene expression as part of a large complex thatcontains corepressors, nuclear receptor corepressor 1 (NCoR) andsilencing mediator for retinoid and thyroid-hormone receptors (SMRT), aswell as class IIa HDACs, such as HDAC4 (Guenther et al. 2000, Genes Dev.14:1048-1057; Li et al., 2000, EMBO J. 19:4342-4350) (for review, seeKaragianni, 2007, Oncogene 26:5439-5449). NCoR associates with HDAC3through the deacetylase activation domain (DAD) of NCoR and a singleamino acid substitution (Y478A) in the NCoR DAD results in a mutantprotein that is unable to associate with or activate HDAC3 (Alenghat etal., 2008, Nature 456:997-1000). In addition, class IIa HDACs mayrequire interaction with HDAC3 for their HDAC activity (Fischle et al.,2002, Mol. Cell 9:45-57). It has been demonstrated that HDAC3 is acritical negative regulator of long-term memory formation. Specifically,focal deletion of HDAC3 as well as selective inhibition of HDAC3significantly enhanced long-term memory in a persistent manner (McQuown,2011, 31(2)764-774).

A “memory” as used herein refers to the ability to recover informationabout past events or knowledge. Memories include short-term memory (alsoreferred to as working or recent memory) and long-term memory.Short-term memories involve recent events, while long-term memoriesrelate to the recall of events of the more distant past. Methods ofassessing the ability to recall a memory are known to those of skill inthe art and may involve routine cognitive tests. Enhancing or retrievingmemories is distinct from learning. However, in some instances in theart learning is referred to as memory. Learning, unlike memoryenhancement, refers to the ability to create new memories that had notpreviously existed. Thus in order to test the ability of a compound toeffect the ability of a subject to learn rather than recall oldmemories, the compound would be administered prior to or at the sametime as the memory is created. In order to test the ability of acompound to affect recall of a previously created memory the compound isadministered after the memory is created and preferably after the memoryis lost.

As used herein “age related memory loss” refers to any of a continuum ofconditions characterized by a deterioration of neurological functioningthat does not rise to the level of a dementia, as further defined hereinand/or as defined by the Diagnostic and Statistical Manual of MentalDisorders: 4th Edition of the American Psychiatric Association (DSM-IV,1994). Age related memory loss is characterized by objective loss ofmemory in an older subject compared to his or her younger years, butcognitive test performance that is within normal limits for thesubject's age. Age related memory loss subjects score within a normalrange on standardized diagnostic tests for dementias, as set forth bythe DSM-IV. Moreover, the DSM-IV provides separate diagnostic criteriafor a condition termed Age-Related Cognitive Decline. In the context ofthe present invention, as well as the terms “Age-Associated MemoryImpairment” and “Age-Consistent Memory Decline” are understood to besynonymous with the age related memory loss. Age-related memory loss mayinclude decreased brain weight, gyral atrophy, ventricular dilation, andselective loss of neurons within different brain regions. For purposesof some embodiments of the present invention, more progressive forms ofmemory loss are also included under the definition of age-related memorydisorder. Thus persons having greater than age-normal memory loss andcognitive impairment, yet scoring below the diagnostic threshold forfrank dementia, may be referred to as having a mild neurocognitivedisorder, mild cognitive impairment, late-life forgetfulness, benignsenescent forgetfulness, incipient dementia, provisional dementia, andthe like. Such subjects may be slightly more susceptible to developingfrank dementia in later life (See also US patent application 2006/008517(Vasogen Ireland limited) which is incorporated by reference). Symptomsassociated with age-related memory loss include but are not limited toalterations in biochemical markers associated with the aging brain, suchas IL-1 beta, IFN-gamma, p-JNK, p-ERK, reduction in synaptic activity orfunction, such as synaptic plasticity, evidenced by reduction in longterm potentiation, diminution of memory and learning.

As used herein “injury related memory loss” refers to a loss of memorywherein there is damage to the brain, and there may have also beenneurological damage. Sources of brain injury include traumatic braininjury such as concussive injuries or penetrating head wounds, braintumors, alcoholism, Alzheimer's disease, stroke, heart attack and otherconditions that deprive the brain of oxygen, meningitis, AIDS, viralencephalitis, and hydrocephalus.

Methods for enhancing memories can include reestablishing access tomemories as well as recapturing memories. The term re-establishingaccess as used herein refers to increasing retrieval of a memory.Although Applicants are not bound by a mechanism of action, it isbelieved that the compounds of the invention are effective in increasingretrieval of memories by re-establishing a synaptic network. The processof re-establishing a synaptic network may include an increase in thenumber of active brain synapses and or a reversal of neuronal loss.

Neurogenesis, or the birth of new neuronal cells, was thought to occuronly in developing organisms. However, recent research has demonstratedthat neurogenesis does indeed continue into and throughout adult life.On going neurogenesis is thought to be an important mechanism underlyingneuronal plasticity, enabling organisms to adapt to environmentalchanges and influencing learning and memory throughout life. In oneaspect, the invention includes a method of increasing synaptic densityin a subject comprising administering to the subject in need of suchincrease a compound of the invention or a pharmaceutically acceptablesalt, hydrate, solvate, or prodrug thereof. In one aspect, the inventionincludes a method of increasing synaptic plasticity in a subjectcomprising administering to the subject in need of such increase acompound of the invention or a pharmaceutically acceptable salt,hydrate, solvate, or prodrug thereof. In one aspect, the inventionincludes a method of increasing dendritic density in neurons in asubject comprising administering to the subject in need of such increasea compound of the invention or a pharmaceutically acceptable salt,hydrate, solvate, or prodrug thereof.

The invention provides methods for enhancing memory in a subject havinga memory disorder. Examples of types of memory disorders includeAlzheimer's disease, absent-minded professor, absent-mindedness,amnesia, anterograde amnesia, blackout (alcohol-related amnesia),bromism, childhood amnesia, false memory syndrome, fugue state,hyperthymesia, Korsakoff's syndrome, lacunar amnesia, memory distrustsyndrome, memory loss, post-traumatic amnesia, prosopamnesia,psychogenic amnesia, repressed memory, retrograde amnesia, Ribot's Law,selective memory loss, sywald skeid, source amnesia, source-monitoringerror, the seven sins of memory, tip of the tongue, transient epilepticamensia, transient global amnesia, and twilight sleep.

In one embodiment, Alzheimer's disease is the memory disorder. Suchmethods optionally involve administering the inhibitor and monitoringthe subject to identify recapture of a memory that was previously lost.Subjects may be monitored by routine tests known in the art.

In other embodiments the alzheimer's subject is one that has late stageAlzheimer' s disease. Many of the drugs suggested for treatingAlzheimer's disease are designed to treat the early stages of thedisease by preventing plaque buildup. The compounds of the invention areuseful for treating both early stages and late stages of dementiabecause they actually improve memory and cognition rather thanpreventing only plaque accumulation.

Cognitive Function Disorders/Impairment

The invention relates to methods of treating, alleviating, and/orpreventing cognitive function disorders/impairments.

Impaired cognitive function refers to cognitive function that is not asrobust as that observed in an age-matched normal subject and includesstates in which cognitive function is reduced. In some cases, cognitivefunction is reduced by about 5%, about 10%, about 30%, or more, comparedto cognitive function measured in an age-matched normal subject.Cognitive function may be promoted to any detectable degree, but inhumans preferably is promoted sufficiently to allow an impaired subjectto carry out daily activities of normal life.

In some embodiments, the cognitive function disorders or impairments areassociated with, but not limited to, Alzheimer's disease, Huntington'sdisease, seizure induced memory loss, schizophrenia, Rubinstein Taybisyndrome, Rett Syndrome, Fragile X, Lewey body dementia, Vasculardementia, bipolar disorder and social, cognitive and learning disordersassociated with autism, attention deficit hyperactivity disorder (ADHD),dyselexia, learning disorders, traumatic head injury, stroke inducedcognitive and motor impairment, traumatic brain injury,neurodegeneration and neuronal loss mediated cognitive impairment, andattention deficit disorder.

In some embodiments, the cognitive function disorders or impairments areassociated with, but not limited to, anxiety disorders, conditioned fearresponse, panic disorders, obsessive compulsive disorders,post-traumatic stress disorder, phobias, social anxiety disorders,substance dependence recovery or Age Associated Memory Impairment(AAMI), and Age Related Cognitive Decline (ARCD).

In some embodiments, the invention relates to methods of treating,alleviating, and/or preventing vascular dementia. Vascular dementia,also referred to as “multi-infarct dementia”, refers to a group ofsyndromes caused by different mechanisms all resulting in vascularlesions in the brain. The main subtypes of vascular dementia are, forexample vascular mild cognitive impairment, multi-infarct dementia,vascular dementia due to a strategic single infarct (affecting thethalamus, the anterior cerebral artery, the parietal lobes or thecingulate gyms), vascular dementia due to hemorrhagic lesions, smallvessel disease (including, e.g. vascular dementia due to lacunar lesionsand Binswanger disease), and mixed Alzheimer's Disease with vasculardementia.

In some embodiments, the invention relates to treating, alleviating,and/or preventing Huntington's Disease. Huntington's Disease is aneurological disease which results in cognitive decline associated withinexorable progression to death. Cognitive symptoms associated withHuntington's Disease include loss of intellectual speed, attention, andshort term memory and/or behavioral symptoms.

Cognitive function may be assessed, and thus optionally defined, via oneor more tests or assays for cognitive function. Non-limiting examples ofa test or assay for cognitive function include CANTAB (see for exampleFray et al. “CANTAB battery: proposed utility in neurotoxicology.”Neurotoxicol Teratol 1996; 18(4):499-504), Stroop Test, Trail Making,Wechsler Digit Span, or the CogState computerized cognitive test (seealso Dehaene et al. “Reward-dependent learning in neuronal networks forplanning and decision making.” Brain Res. 2000; 126:21729; Iverson etal. “Interpreting change on the WAIS-III/WMS-III in clinical samples.”Arch Clin Neuropsychol. 2001; 16(2):183-91; and Weaver et al. “Mildmemory impairment in healthy older adults is distinct from normalaging.” Cogn. 2006; 60(2): 146-55). The methods of the invention may beused to promote cognitive function in a normal subject or to treat,alleviate and/or prevent a subject from having a cognitive dysfunction.A normal subject, as used herein, is a subject that has not beendiagnosed with a disorder associated with impaired cognitive function.

Extinction Learning Disorders

In one aspect, the invention relates to methods of treating,alleviating, and/or preventing extinction learning disorders e.g., afear extinction deficit.

It has been demonstrated that administration of the HDAC inhibitorssodium butyrate or trichostatin A facilitates fear extinction in miceand this enhancement mirrors that caused by commonly used behavioralmanipulation and is consistent with other studies demonstrating a rolefor the hippocampus in the extinction of contextual fear (Lattal, etal., 2007, Behav. Neurosci. 121, 5, 1125-1131).

Compounds of the invention can be used to facilitate the psychologicalprocess of extinction learning and thus are useful for treating,alleviating, and/or preventing neuropsychiatric disorders and otherrelated disorders. Unlike traditional anti-anxiety drugs that areadministered on a chronic basis and address physiological symptoms ofanxiety, the compounds of the invention can be used on a chronic oracute basis in conjunction with a second therapy e.g., psychotherapy.

In one aspect, the present invention is directed to methods fortreating, alleviating, and/or preventing a subject from having aneuropsychiatric disorder. The methods comprise subjecting the subjectto one or more sessions of a combination therapy protocol, where thecombination therapy protocol comprises an acute administration of atherapeutically effective amount of a compound of the invention thatenhances learning or conditioning in combination with a session ofpsychotherapy. By “acute administration” is intended a single exposureof the subject to the therapeutically effective amount of the compoundthat enhances learning or conditioning. In one aspect, the exposure tothe compound occurs within about 24 hours prior to initiating thesession of psychotherapy, preferably within about 12 hours, and morepreferably within about 6 hours prior to initiating the session ofpsychotherapy. A full course of treatment for the neuropsychiatricdisorder entails at least one session of this combination therapyprotocol.

For purposes of the present invention, a subject may have a singledisorder, or may have a constellation of disorders that are to betreated, alleviated, and/or prevented by the methods described herein.

The neuropsychiatric disorders contemplated in the present inventioninclude, but are not limited to, fear and anxiety disorders, addictivedisorders including substance-abuse disorders, and mood disorders.Within the fear and anxiety disorder category, the invention encompassesthe treatment or prevention of panic disorder, specific phobia,post-traumatic stress disorder (PTSD), obsessive-compulsive disorder,and movement disorders such as Tourette's syndrome. The disorderscontemplated herein are defined in, for example, the DSM-IV (Diagnosticand Statistical Manual of Mental Disorders (4th ed., AmericanPsychiatric Association, Washington D.C., 1994)), which is hereinincorporated by reference.

Anxiety-related disorders relate to those disorders characterized byfear, anxiety, addiction, and the like. Patients with anxiety-relateddisorders can have a single such disorder, or can have a constellationof disorders. The anxiety-related disorders contemplated in the presentinvention include, but are not limited to, anxiety disorders, addictivedisorders including substance-abuse disorders, mood disorders (e.g.,depression and/or bipolar disorder), movement disorders such asTourette's syndrome, psychogenic erectile dysfunction (impotenceresulting from a man's inability to obtain or maintain an erection ofhis penis), insomnia (e.g. chronic insomnia), and eating disorders (e.g.anorexia).

Anxiety disorders include, but are not limited to, panic disorder,agoraphobia, social phobia, specific phobia, PTSD, obsessive-compulsivedisorder, and generalized anxiety disorder. The disorders contemplatedherein are defined in, for example, the DSM-IV (Diagnostic andStatistical Manual of Mental Disorders (4th ed., American PsychiatricAssociation, Washington D.C., 1994)).

Movement disorders are neurological conditions that affect the speed,fluency, quality, and ease of movement. Representative movementdisorders include but are not limited to ataxia, chorea, myoclonus,dystonia, Parkinson's disease, restless leg syndrome, tics, andTourette's syndrome. Movement disorders typically occur as a result ofdamage or disease in the basal ganglia region of the brain. Movementdisorders can result from age-related changes, medications, geneticdisorders, metabolic disorders, disease, stroke, or injury. Recovery ofmovement after stroke or injury may be facilitated when treatedaccording to the methods of the invention.

Addictive disorders are disorders characterized by addiction to anactivity or substance, and include, for example, alcohol addiction, drugaddiction, and gambling addiction.

Depression refers to the clinical condition known as major depressivedisorder, and is characterized by a state of intense sadness,melancholia, or despair that has advanced to the point of beingdisruptive to an individual's social functioning and/or activities ofdaily living. Depression is alleviated if either (or both) the severityor frequency of a symptom of the depression is reduced. However, asubject can be treated for depression in accordance with the methods ofthe invention irrespective of whether the treatment actually wassuccessful in alleviating the depression.

Insomnia is defined herein as the inability to fall asleep or to stayasleep for a sufficient amount of time during regular sleeping hours. Itincludes acute insomnia, which occurs in either a transient or shortterm form, and chronic insomnia. It also includes initial insomnia,defined as difficulty in falling asleep; middle insomnia, defined asawakening in the middle of the night followed by eventually falling backto sleep, but with difficulty; and terminal insomnia, defined asawakening before one's usual waking time and being unable to return tosleep.

As defined by the National Institute of Mental Health, Autism SpectrumDisorders (ASD), also widely known as Pervasive Developmental Disorders(PDDs), cause severe and pervasive impairment in thinking, feeling,language, and the ability to relate to others. These disorders areusually first diagnosed in early childhood and range from a severe form,called autistic disorder, through pervasive development disorder nototherwise specified (PDD-NOS), to a much milder form, Asperger syndrome.They also include two rare disorders, Rett syndrome and childhooddisintegrative disorder.

Attention-Deficit Hyperactivity Disorder (ADHD) is one of the mostcommon mental disorders that develop in children. Children with ADHDtypically have impaired functioning in multiple settings, includinghome, school, and in relationships with peers. Symptoms of ADHD includeimpulsiveness, hyperactivity, and inattention.

Typical treatments encompassed by the present invention includecombination therapies. For instance, the combination therapy may be apharmacotherapy (i.e., a compound of the invention) and a behavioraltherapy. Behavioral therapy comprises, but is not limited to,electroconvulsive seizure therapy, exercise, group therapy, talktherapy, or conditioning. In another embodiment, the behavioral therapyis cognitive-behavioral therapy. Examples of behavioral therapy that maybe used in the ongoing methods are described, for example, inCognitive-Behavioral Therapies by K. Dobson, ed.., GuilfordPublications, Inc., 2002; The new Handbook of Cognitive Therapy: Basicsand Beyond by Judith S. S. Beck, Guilford Publications, Inc. 1995 hereinincorporated by reference in their entireties. Any pharmaceutical activeingredient that is recognized by the skilled artisan as being apharmacologic agent that enhances learning or conditioning can be usedin the methods of the invention. For example, one such class ofpharmaceutical active ingredients contemplated herein comprisescompounds that increase the level of norepinephrine in the brain. Suchcompounds include those acting as norepinephrine reuptake inhibitors,for example tomoxetine, reboxetine, duloxetine, venlafaxine, andmilnacipran, and those compounds that cause release of norepinephrine,for example amphetamine, dextroamphetamine, pemoline, andmethylphenidate. Another class of such pharmaceutical active ingredientsis those compounds that increase the level of acetylcholine in thebrain, including, for example, compounds that block its breakdown.Examples of such compounds include, but are not limited to, donepezilHCl or Aricept™ and tacrine, which inhibit cholinesterase activity.

Methods of the invention also encompass the use in combination with acompound of the invention of any type of psychotherapy that is suitablefor the particular psychiatric disorder for which the subject isundergoing treatment. Suitable methods of psychotherapy include exposurebased psychotherapy, cognitive psychotherapy, and psychodynamicallyoriented psychotherapy. Methods of the invention also encompass exposingthe subject to cognitive behavioral therapy (CBT), behavioral exposuretreatments, virtual reality exposure (VRE) or cognitive remediationtherapy.

Methods of the invention also encompass extinction training. The goal ofextinction training is to pair a stimulus that previously provoked adeleterious, unwanted response with a new learning that will not lead toa negative outcome, thereby generating in a subject a new, moreappropriate response to the stimulus to compete with and ideally replacethe previous undesirable response. Extinction training frequentlyexposes a subject to a stimulus or situation in the absence of anaversive consequence, e.g., a subject that has deleterious, high anxietyresponses to a given stimulus or situation is exposed to that stimulusor situation in the absence of an aversive consequence. A typical goalof extinction training is to produce new learning in the subject thatresults from the pairing of the original stimulus or situation with anon-deleterious outcome, thereby generating, in subsequent exposures tothe stimulus, a more appropriate response in place of the unwantedresponse. An extinction learning event refers to a completedstimulus/response extinction training cycle.

One form of extinction training entails psychotherapy. For example, themethods of the invention contemplate treating, alleviating, and/orpreventing anxiety disorders by: (i) administering psychotherapy totreat, alleviate, and/or prevent an anxiety-related disorder in asuitable human subject, and (ii) administering a therapeuticallyeffective dose a compound of the invention to said subject on anachronic, post-training, pre-sleep basis. Suitable methods ofpsychotherapy include but are not limited to exposure-basedpsychotherapy, cognitive psychotherapy, and psychodynamically orientedpsychotherapy.

One method of psychotherapy that is specifically contemplated is the useof virtual reality (VR) exposure therapy to treat, alleviate, and/orprevent an anxiety disorder using the methods of the invention.

Another method of psychotherapy that is particularly beneficial whenutilized in combination with a compound or composition of the presentinvention is cognitive behavioral therapy (“CBT”). CBT is a form ofpsychotherapy that combines cognitive therapy and behavior therapy, andemphasizes the critical role of thinking in causing people to act andfeel as they do. Therefore, if an individual is experiencing unwantedfeelings and behaviors, CBT teaches that it is important to identify thethinking that is causing the undesirable feelings and/or behaviors andto learn how to replace this deleterious thinking with thoughts thatlead to more desirable reactions. CBT is widely used to help people whoare experiencing a range of mental health difficulties, some of which donot conveniently fit definitions of a particular medical affliction. CBThas been used to treat anxiety disorders, mood disorders, addictivedisorders, eating disorders, insomnia, chronic pain, schizophrenia,fibromyalgia, ADHD, and autism spectrum disorders, among others.Post-extinction training pre-sleep administration of a compound of theinvention, subsequent to CBT treatment, can be used to augment theeffectiveness of the CBT treatment for these medical conditions.

In one embodiment, subjects suffering from social anxiety disorderundergo weekly cognitive behavioral therapy sessions to treat theaffliction. After each therapy session, subjects are administered atherapeutically effective formulation of compounds of the invention on apost-extinction training pre-sleep basis. Relative to subjects treatedonly via cognitive behavioral therapy, or to subjects treated viacognitive behavioral therapy and a placebo, anxiety associated withsocial anxiety disorder is expected to be reduced to a greater extent insubjects treated with a combination of cognitive behavioral therapy andachronic administration of a compound of the invention on apost-extinction training pre-sleep basis.

In another embodiment of the invention, a compound of the invention isadministered after extinction training only if the extinction trainingyields positive results on that day. For example, a subject undergoingcognitive behavioral therapy for PTSD is administered a compound of theinvention on a post-extinction training only if the cognitive behavioraltherapy was deemed to be successful, as determined by the subject and/ortherapist. In one aspect, the compound is administered on apost-extinction, pre-sleep basis. In another aspect, a subjectundergoing cognitive behavioral therapy for PTSD is administered acompound of the invention on a pre-extinction training. In one aspect,the compound is administered on a pre-extinction, pre-sleep basis. Thismethod may also be useful when applied to treatment of autism spectrumdisorders or attention-deficit hyperactivity disorder.

In another embodiment of the invention, subjects afflicted with anxietydisorders such as PTSD receive extinction training using Eye MovementDesensitization and Reprocessing (EMDR), and subsequently areadministered a therapeutically effective dose of a compound of theinvention on a post-extinction training pre-sleep basis. Another form ofextinction training is provided by biofeedback, which is particularlyuseful in enabling subjects to learn to control physiological processesthat normally occur involuntarily, such as blood pressure, heart rate,muscle tension, and skin temperature. As used herein, “biofeedback”refers to a technique in which subjects are trained to improve theirhealth by using signals from their own bodies to control their ownphysiological responses.

In one embodiment of the invention, a subject suffering from chronicpain undergoes biofeedback sessions to help alleviate the pain. Upon theconclusion of each session wherein the subject has made progress inlearning/developing responses that reduce the chronic pain, the subjectis administered a compound of the invention on a post-extinctiontraining pre-sleep basis in order to consolidate the desired learning.

In another embodiment, a subject suffering from phantom limb syndromeundergoes thermal biofeedback sessions to reduce and hopefully eliminatethe symptoms. After each session, the subject is administered atherapeutically effective formulation of a compound of the invention ona post-extinction training pre-sleep basis.

In another embodiment, extinction training can be provided by physicaltherapy, or virtual reality physical therapy such as virtual realitygait therapy. For example, a stroke victim re-learning how to walk canundergo virtual reality gait therapy, and then be administered acompound of the invention on an achronic, post-extinction trainingpre-sleep basis.

Another form of extinction training can be provided by pharmacotherapy.For example, a man afflicted with erectile dysfunction can have anextinction learning event based on a positive sexual outcome, includinginstances wherein the positive sexual outcome was achieved with thepharmacological assistance of a PDE-5 inhibitor such as sildenafil,tadalafil, vardenafil, and/or udenafil. By administering a compound ofthe invention on a post-extinction training pre-sleep basis to a subjectwith erectile dysfunction, following a successful sexual outcome whereinthe subject utilized sildenafil, the heightened confidence and reducedsexual performance anxiety resulting from a successful outcome can beconsolidated in said subject's psyche, thereby facilitating extinctionof any deleterious performance anxiety associated with sexualintercourse.

Extinction training does not always require intervention of a trainedspecialist. Individuals can carry out extinction training on themselves.

Fungal Diseases or Infections

In some aspects, the invention relates to a method for treating,alleviating, and/or preventing a fungal disease or infection comprisingadministering to a subject a compound of the invention. The inventionprovides a method for treating, alleviating, and/or preventing ahospital-acquired fungal infections that attack immunocompromisedpatients including those with HIV and cancer. In one embodiment, theinvention provides a method for treating, alleviating, and/or preventinga fungal disease in a subject not suffering from cancer.

Inflammatory Disease

In some aspects, the invention relates to a method for treating,alleviating, and/or preventing an inflammatory disease, including butnot limited to stroke, rheumatoid arthritis, lupus erythematosus,ulcerative colitis and traumatic brain injuries (Leoni et al., PNAS,99(5); 2995-3000(2002); Suuronen et al. J. Neurochem. 87; 407-416 (2003)and Drug Discovery Today, 10: 197-204 (2005).

Neoplastic Diseases

In some aspects, the invention relates to methods of selectivelyinducing terminal differentiation, and arresting cell growth and/orapoptosis of neoplastic cells, thereby inhibiting proliferation of suchcells. The compounds of the present invention are useful in treating,alleviating, and/or preventing cancer in a subject.

The term “cancer” refers to any cancer caused by the proliferation ofneoplastic cells, such as solid tumors, neoplasms, carcinomas, sarcomas,leukemias, lymphomas and the like. In particular, cancers that may betreated, alleviated and/or prevented by the compounds of the inventioninclude, but are not limited to: cardiac cancer, lung cancer,gastrointestinal cancer, genitourinary tract cancer, liver cancer,nervous system cancer, gynecological cancer, hematologic cancer, skincancer, and adrenal gland cancer.

In some embodiments, the compounds of the invention relate to treating,alleviating, or preventing cardiac cancers selected from sarcoma(angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma,rhabdomyoma, fibroma, lipoma and teratoma.

In some embodiments, the compounds of the invention relate to treating,alleviating, or preventing lung cancer selected from bronchogeniccarcinoma (squamous cell, undifferentiated small cell, undifferentiatedlarge cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchialadenoma, sarcoma, lymphoma, chondromatous hamartoma, and mesothelioma.

In some embodiments, the compounds of the invention relate to treating,alleviating or preventing gastrointestinal cancer selected fromesophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma,lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas(ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoidtumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoidtumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma,fibroma), and large bowel (adenocarcinoma, tubular adenoma, villousadenoma, hamartoma, leiomyoma).

In some embodiments, the compounds of the invention relate to treating,alleviating, and/or preventing genitourinary tract cancer selected fromkidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma,leukemia), bladder and urethra (squamous cell carcinoma, transitionalcell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), andtestis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma,choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma,fibroadenoma, adenomatoid tumors, lipoma).

In some embodiments, the compounds of the invention relate to treating,alleviating, and/or preventing liver cancer selected from hepatoma(hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, and hemangioma.

In some embodiments, the compounds of the invention relate to treating,alleviating, and/or preventing bone cancer selected from osteogenicsarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cellsarcoma), multiple myeloma, malignant giant cell tumor chordoma,osteochronfroma (osteocartilaginous exostoses), benign chondroma,chondroblastoma, chondromyxofibroma, osteoid osteoma and giant celltumors.

In some embodiments, the compounds of the invention relate to treating,alleviating, and/or preventing nervous system cancer selected from skull(osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges(meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma,medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastomamultiform, oligodendroglioma, schwannoma, retinoblastoma, congenitaltumors), and spinal cord (neurofibroma, meningioma, glioma, sarcoma).

In some embodiments, the compounds of the invention relate to treating,alleviating, and/or preventing gynecological cancer selected from uterus(endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervicaldysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma,mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecalcell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignantteratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma,adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma,squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma),and fallopian tubes (carcinoma).

In some embodiments, the compounds of the invention relate to treating,alleviating, and/or preventing skin cancer selected from malignantmelanoma, basal cell carcinoma, squamous cell carcinoma, Karposi'ssarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma,keloids, and psoriasis.

In some embodiments, the compounds of the invention relate to methods oftreating, alleviating, and/or preventing adrenal gland cancer selectedfrom neuroblastoma.

In some embodiments, the instant compounds are useful in the treatment,alleviation, and/or preventing of cancers that include, but are notlimited to: leukemias including acute leukemias and chronic leukemiassuch as acute lymphocytic leukemia (ALL), Acute myeloid leukemia (AML),chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML)and Hairy Cell Leukemia; lymphomas such as cutaneous T-cell lymphomas(CTCL), noncutaneous peripheral T-cell lymphomas, lymphomas associatedwith human T-cell lymphotrophic virus (HTLV) such as adult T-cellleukemia/lymphoma (ATLL), Hodgkin's disease and non-Hodgkin's lymphomas,large-cell lymphomas, diffuse large B-cell lymphoma (DLBCL); Burkitt'slymphoma; mesothelioma, primary central nervous system (CNS) lymphoma;multiple myeloma; childhood solid tumors such as brain tumors,neuroblastoma, retinoblastoma, Wilm's tumor, bone tumors, andsoft-tissue sarcomas, common solid tumors of adults such as head andneck cancers (e.g., oral, laryngeal and esophageal), genito urinarycancers (e.g., prostate, bladder, renal, uterine, ovarian, testicular,rectal and colon), lung cancer, breast cancer, pancreatic cancer,melanoma and other skin cancers, stomach cancer, brain tumors, livercancer and thyroid cancer.

Hematologic Diseases

In some aspects, the invention relates to methods of treating,alleviating, or preventing hematolical diseases. Hematologic diseasesinclude abnormal growth of blood cells which can lead to dysplasticchanges in blood cells and hematologic malignancies such as variousleukemias. Examples of hematologic diseases include but are not limitedto acute myeloid leukemia, acute promyelocytic leukemia, acutelymphoblastic leukemia, chronic myelogenous leukemia, themyelodysplastic syndromes, and sickle cell anemia.

Acute myeloid leukemia (AML) is the most common type of acute leukemiathat occurs in adults. Several inherited genetic disorders andimmunodeficiency states are associated with an increased risk of AML.These include disorders with defects in DNA stability, leading to randomchormosomal breakage, such as Bloom's syndrome, Fanconi's anemia,Li-Fraumeni kindreds, ataxia-telangiectasia, and X-linkedagammaglobulinemia.

Acute promyelocytic leukemia (APML) represents a distinct subgroup ofAML. This subtype is characterized by promyelocytic blasts containingthe 15; 17 chromosomal translocation. This translocation leads to thegeneration of the fusion transcript comprised of the retinoic acidreceptor and a sequence PML.

Acute lymphoblastic leukemia (ALL) is a heterogenerous disease withdistinct clinical features displayed by various subtypes. Reoccurringcytogenetic abnormalities have been demonstrated in ALL. The most commoncytogenetic abnormality is the 9;22 translocation. The resultantPhiladelphia chromosome represents poor prognosis of the patient.

Chronic myelogenous leukemia (CML) is a clonal myeloproliferativedisorder of a pluripotent stem cell. CML is characterized by a specificchromosomal abnormality involving the translocation of chromosomes 9 and22, creating the Philadelphia chromosome. Ionizing radiation isassociated with the development of CML.

The myelodysplastic syndromes (MDS) are heterogeneous clonalhematopoietic stem cell disorders grouped together because of thepresence of dysplastic changes in one or more of the hematopoieticlineages including dysplastic changes in the myeloid, erythroid, andmegakaryocytic series. These changes result in cytopenias in one or moreof the three lineages. Patients afflicted with MDS typically developcomplications related to anemia, neutropenia (infections), orthrombocytopenia (bleeding). Generally, from about 10% to about 70% ofpatients with MDS develop acute leukemia.

Sickle cell disease is attributable to homozygous inheritance of asingle amino acid substitution in the β-globin gene that leads topolymerization of deoxygenated hemoglobin, deformation of red bloodcells, microvascular occlusion, hemolysis, and consequent diseasemanifestations, including pain, strokes, and pulmonary complications(Bunn H F, 1997, J. Med. 337:762-769). Abundant biochemical,epidemiological, and clinical evidence have shown that a high level of γglobin, the fetal form of β globin, inhibits the aberrant polymerizationof sickle hemoglobin and ameliorates the disease phenotype. The onlyFood and Drug Administration (FDA)-approved drug for sickle celldisease, hydroxyurea, causes significant induction of fetal hemoglobin,decreased disease severity, and benefits overall mortality (Letvin etal., 1984, N Engl J Med 310:869-873; Platt O S, et al., 1984, J ClinInvest 74:652-656; Charache S, et al., 1995, N Engl J. Med 332:317-1322; Steinberg M H, et al., 2003, JAMA 289:1645-1651).Nevertheless, hydroxyurea has bone marrow-suppressive effects and isineffective in a significant portion of patients (Charache S, et al.;Steinberg M H, et al., 2003; Steinberg M H, 1999, N Engl J. Med340:1021-1030). A drug that induces fetal hemoglobin more substantiallywith less myelosuppression would be expected to have greater therapeuticutility in sickle cell disease.

Transcriptional regulation of the human globin gene locus has beeninvestigated intensively. Gamma-globin gene expression is influenced bytranscription factors (GATA-1, EKLF, NF-E4p22, Ikaros) and chromatinmodifying enzymes [SWI/SNF complex, HATs, and histone deacetylase(HDACs)] as part of multiprotein complexes, and a unique, dynamicchromatin structure termed the β-globin active chromatin hub (βACH)(8-11). Polymorphisms in BCL11A, a transcriptional repressor, alterbaseline fetal hemoglobin levels, and a multiprotein complex containingBCL11a binds to the β-globin locus, resulting in repression of γ-globinexpression (Menzel S, et al., 2007, Nat Genet 39:1197-1199; Lettre G, etal., 2008, Proc Natl Acad Sci USA 105:11869-11874; Sankaran V G, et al.,2008, Science 322:1839-1842; Uda M, et al., 2008, Proc NATL Acad Sci USA105:1620-1625; Sankaran V G, et al., 2009, Nature 460:1093-1097).Despite this granularity, discrete targets amenable to ligand discoveryefforts have not been identified and functionally validated.

The induction of fetal hemoglobin is a validated strategy to improvesymptoms and complications of sickle cell disease. The development oftargeted therapies has been limited by the absence of discrete druggabletargets. Bradner et al., 2010, PNAS, 107:28, 12617-12622 has developed aunique bead-based strategy for the identification of inducers of fetalhemoglobin transcripts in primary human erythroid cells, which includesa small-molecule screen of bioactive compounds that have been identifiedto have remarkable class-associated activity among histone deacetylase(HDAC) inhibitors. Using a chemical genetic strategy combining focusedlibraries of biased chemical probes and reverse genetics by RNAinterference, Bradner et al. identified HDAC1 and HDAC2 as moleculartargets mediating fetal hemoglobin induction. Isoform-selectiveinhibitors of HDAC1 and HDAC2 are targets for the treatment of sicklecell disease.

Formulations

The compounds of the invention may be administered alone (e.g., insaline or buffer) or using any delivery vehicles known in the art. Forinstance the following delivery vehicles have been described:Cochleates; Emulsomes, ISCOMs; Liposomes; Live bacterial vectors (e.g.,Salmonella, Escherichia coli, Bacillus calmatte-guerin, Shigella,Lactobacillus); Live viral vectors (e.g., Vaccinia, adenovirus, HerpesSimplex); Microspheres; Nucleic acid vaccines; Polymers; Polymer rings;Proteosomes; Sodium Fluoride; Transgenic plants; Virosomes; Virus-likeparticles. Other delivery vehicles are known in the art and someadditional examples are provided below.

The term an “effective amount” of a compound of the invention refers tothe amount necessary or sufficient to realize a desired biologic effect.For example, an effective amount of a compound of the invention is thatamount sufficient to treat a condition. In another aspect, an effectiveamount of a compound is that amount sufficient to alleviate a condition.In another aspect, an effective amount of a compound is that amountsufficient to prevent a condition. Combined with the teachings providedherein, by choosing among the various active compounds and weighingfactors such as potency, relative bioavailability, patient body weight,severity of adverse side-effects and preferred mode of administration,an effective prophylactic or therapeutic treatment regimen can beplanned which does not cause substantial toxicity and yet is entirelyeffective to treat the particular subject. The effective amount for anyparticular application can vary depending on such factors as thecondition being treated, the particular compounds being administered thesize of the subject, or the severity of the condition.

The compounds of the invention may be administered by any route known,such as, for example, orally, transdermally, intravenously, cutaneously,subcutaneously, nasally, intramuscularly, intraperitoneally,intracranially, and intracerebroventricularly.

In certain embodiments, compounds of the invention are administered atdosage levels greater than about 0.001 mg/kg, such as greater than about0.01 mg/kg or greater than about 0.1 mg/kg. For example, the dosagelevel may be from about 0.001 mg/kg to about 50 mg/kg such as from about0.01 mg/kg to about 25 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, orfrom about 1 mg/kg to about 5 mg/kg of subject body weight per day, oneor more times a day, to obtain the desired therapeutic effect. It willalso be appreciated that dosages smaller than 0.001 mg/kg or greaterthan 50 mg/kg (for example 50-100 mg/kg) can also be administered to asubject.

In one embodiment, the compound of the invention is administeredonce-daily, twice-daily, or three-times daily. In one embodiment, thecompound of the invention is administered continuously (i.e., every day)or intermittently (e.g., 3-5 days a week). In another embodiment,administration could be on an intermittent schedule.

Further, administration less frequently than daily, such as, forexample, every other day may be chosen. In additional embodiments,administration with at least 2 days between doses may be chosen. By wayof example only, dosing may be every third day, bi-weekly or weekly. Asanother example, a single, acute dose may be administered.Alternatively, compounds of the invention can be administered on anon-regular basis e.g., whenever symptoms begin. For any compounddescribed herein the effective amount can be initially determined fromanimal models.

Toxicity and efficacy of the compounds of the invention can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Compounds that exhibit large therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

Data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage of the compounds of the inventionfor use in humans. The dosage of such agents lies preferably within arange of circulating concentrations that include the ED50 with little orno toxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the invention, the effective dose can beestimated initially from cell culture assays. A dose may be formulatedin animal models to achieve a circulating plasma concentration rangethat includes the IC50 (i.e., the concentration of the test compoundthat achieves a half-maximal inhibition of symptoms) as determined incell culture. Such information can be used to more accurately determineuseful doses in humans. Levels in plasma may be measured, for example,by high performance liquid chromatography. In certain embodiments,pharmaceutical compositions may comprise, for example, at least about0.1% of an active compound. In other embodiments, the an active compoundmay comprise between about 2% to about 75% of the weight of the unit, orbetween about 25% to about 60%, for example, and any range derivabletherein. Multiple doses of the compounds of the invention are alsocontemplated.

The formulations of the invention are administered in pharmaceuticallyacceptable solutions, which may routinely contain pharmaceuticallyacceptable concentrations of salt, buffering agents, preservatives,compatible carriers, and optionally other therapeutic ingredients.

For use in therapy, an effective amount of one or more compounds of theinvention can be administered to a subject by any mode that delivers thecompound(s) to the desired surface, e.g., mucosal, systemic.Administering the pharmaceutical composition of the present inventionmay be accomplished by any means known to the skilled artisan. Compoundsof the invention may be administered orally, transdermally,intravenously, cutaneously, subcutaneously, nasally, intramuscularly,intraperitoneally, intracranially, or intracerebroventricularly.

For oral administration, one or more compounds of the invention can beformulated readily by combining the active compound(s) withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the compounds of the invention to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a subject to be treated.

Pharmaceutical preparations for oral use can be obtained as solidexcipient, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate. Optionally the oral formulations may also be formulated insaline or buffers, i.e. EDTA for neutralizing internal acid conditionsor may be administered without any carriers.

Also specifically contemplated are oral dosage forms of one or morecompounds of the invention. The compound(s) may be chemically modifiedso that oral delivery of the derivative is efficacious. Generally, thechemical modification contemplated is the attachment of at least onemoiety to the compound itself, where said moiety permits (a) inhibitionof proteolysis; and (b) uptake into the blood stream from the stomach orintestine. Also desired is the increase in overall stability of thecompound(s) and increase in circulation time in the body. Examples ofsuch moieties include: polyethylene glycol, copolymers of ethyleneglycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone and polyproline. Abuchowski and Davis,1981, “Soluble Polymer-Enzyme Adducts” In: Enzymes as Drugs, Hocenbergand Roberts, eds., Wiley-Interscience, New York, N.Y., pp. 367-383;Newmark, et al., 1982, J. Appl. Biochem. 4: 185-189. Other polymers thatcould be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferredfor pharmaceutical usage, as indicated above, are polyethylene glycolmoieties.

The location of release may be the stomach, the small intestine (theduodenum, the jejunum, or the ileum), or the large intestine. Oneskilled in the art has available formulations which will not dissolve inthe stomach, yet will release the material in the duodenum or elsewherein the intestine. Preferably, the release will avoid the deleteriouseffects of the stomach environment, either by protection of the compoundor by release of the biologically active material beyond the stomachenvironment, such as in the intestine.

To ensure full gastric resistance a coating impermeable to at least pH5.0 is important. Examples of the more common inert ingredients that areused as enteric coatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55,polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, celluloseacetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. Thesecoatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which arenot intended for protection against the stomach. This can include sugarcoatings, or coatings which make the tablet easier to swallow. Capsulesmay consist of a hard shell (such as gelatin) for delivery of drytherapeutic i.e. powder; for liquid forms, a soft gelatin shell may beused. The shell material of cachets could be thick starch or otheredible paper. For pills, lozenges, molded tablets or tablet triturates,moist massing techniques can be used.

The compound of the invention can be included in the formulation as finemultiparticulates in the form of granules or pellets of particle sizeabout 1 mm. The formulation of the material for capsule administrationcould also be as a powder, lightly compressed plugs or even as tablets.The compound of the invention could be prepared by compression.

Colorants and flavoring agents may all be included. For example, thecompound of the invention may be formulated (such as by liposome ormicrosphere encapsulation) and then further contained within an edibleproduct, such as a refrigerated beverage containing colorants andflavoring agents.

One may dilute or increase the volume of compound delivered with aninert material. These diluents could include carbohydrates, especiallymannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modifieddextrans and starch. Certain inorganic salts may be also be used asfillers including calcium triphosphate, magnesium carbonate and sodiumchloride. Some commercially available diluents are Fast-Flo, Emdex,STA-Rx 1500, Emcompress and Avicell. Disintegrants may be included inthe formulation of the therapeutic into a solid dosage form. Materialsused as disintegrates include but are not limited to starch, includingthe commercial disintegrant based on starch, Explotab. Sodium starchglycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin,sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose,natural sponge and bentonite may all be used. Another form of thedisintegrants is the insoluble cationic exchange resins. Powdered gumsmay be used as disintegrants and as binders and these can includepowdered gums such as agar, Karaya or tragacanth. Alginic acid and itssodium salt are also useful as disintegrants.

Binders may be used to hold the therapeutic together to form a hardtablet and include materials from natural products such as acacia,tragacanth, starch and gelatin. Others include methyl cellulose (MC),ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both beused in alcoholic solutions to granulate the therapeutic.

An anti-frictional agent may be included in the formulation of thecompound of the invention to prevent sticking during the formulationprocess. Lubricants may be used as a layer between the compound and thedie wall, and these can include but are not limited to; stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricantsmay also be used such as sodium lauryl sulfate, magnesium laurylsulfate, polyethylene glycol of various molecular weights, Carbowax 4000and 6000. Glidants that might improve the flow properties of the drugduring formulation and to aid rearrangement during compression might beadded. The glidants may include starch, talc, pyrogenic silica andhydrated silicoaluminate.

To aid dissolution of the compound into the aqueous environment asurfactant might be added as a wetting agent. Surfactants may includeanionic detergents such as sodium lauryl sulfate, dioctyl sodiumsulfosuccinate and dioctyl sodium sulfonate. Cationic detergents mightbe used and could include benzalkonium chloride or benzethomiumchloride. The list of potential non-ionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the compound eitheralone or as a mixture in different ratios.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Microspheres formulatedfor oral administration may also be used. Such microspheres have beenwell defined in the art. All formulations for oral administration shouldbe in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

Also contemplated herein is pulmonary delivery of the compounds of theinvention. The compound is delivered to the lungs of a mammal whileinhaling and traverses across the lung epithelial lining to the bloodstream. Other reports of inhaled molecules include Adjei et al., 1990,Pharmaceutical Research, 7:565-569; Adjei et al., 1990, InternationalJournal of Pharmaceutics, 63: 135-144 (leuprolide acetate); Braquet etal., 1989, Journal of Cardiovascular Pharmacology, 13(suppl. 5): 143-146(endothelin-1); Hubbard et al., 1989, Annals of Internal Medicine, Vol.IJJ, pp. 206-212 (al-antitrypsin);

Smith et al., 1989, J. Clin. Invest. 84: 1 145-1 146 (a-1-proteinase);Oswein et al., 1990, “Aerosolization of Proteins”, Proceedings ofSymposium on Respiratory Drug Delivery II, Keystone, Colo., March,(recombinant human growth hormone); Debs et al., 1988, J. Immunol.140:3482-3488 (interferon-g and tumor necrosis factor alpha) and Platzet al., U.S. Pat. No. 5,284,656 (granulocyte colony stimulating factor).A method and composition for pulmonary delivery of drugs for systemiceffect is described in U.S. Pat. No. 5,451,569, issued Sep. 19, 1995 toWong et al.

Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art. Some specific examples of commercially availabledevices suitable for the practice of this invention are the Ultraventnebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the AcornII nebulizer, manufactured by Marquest Medical Products, Englewood,Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo Inc.,Research Triangle Park, N.C.; and the Spinhaler powder inhaler,manufactured by Fisons Corp., Bedford, Mass.

All such devices require the use of formulations suitable for thedispensing of compound. Typically, each formulation is specific to thetype of device employed and may involve the use of an appropriatepropellant material, in addition to the usual diluents, and/or carriersuseful in therapy. Also, the use of liposomes, microcapsules ormicrospheres, inclusion complexes, or other types of carriers iscontemplated. Chemically modified compound may also be prepared indifferent formulations depending on the type of chemical modification orthe type of device employed. Formulations suitable for use with anebulizer, either jet or ultrasonic, will typically comprise compounddissolved in water at a concentration of about 0.1 to 25 mg ofbiologically active compound per mL of solution. The formulation mayalso include a buffer and a simple sugar (e.g., for stabilization andregulation of osmotic pressure). The nebulizer formulation may alsocontain a surfactant, to reduce or prevent surface induced aggregationof the compound caused by atomization of the solution in forming theaerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the compound suspended in apropellant with the aid of a surfactant. The propellant may be anyconventional material employed for this purpose, such as achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifiuoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing compound and may also include abulking agent, such as lactose, sorbitol, sucrose, or mannitol inamounts which facilitate dispersal of the powder from the device, e.g.,50 to 90% by weight of the formulation. The compound should mostadvantageously be prepared in particulate form with an average particlesize of less than 10 mm (or microns), most preferably 0.5 to 5 mm, formost effective delivery to the distal lung.

Nasal delivery of a compound of the invention is also contemplated.Nasal delivery allows the passage of a compound of the present inventionto the blood stream directly after administering the therapeutic productto the nose, without the necessity for deposition of the product in thelung. Formulations for nasal delivery include those with dextran orcyclodextran.

For nasal administration, a useful device is a small, hard bottle towhich a metered dose sprayer is attached. In one embodiment, the metereddose is delivered by drawing the pharmaceutical composition of thepresent invention solution into a chamber of defined volume, whichchamber has an aperture dimensioned to aerosolize and aerosolformulation by forming a spray when a liquid in the chamber iscompressed. The chamber is compressed to administer the pharmaceuticalcomposition of the present invention. In a specific embodiment, thechamber is a piston arrangement. Such devices are commerciallyavailable.

Alternatively, a plastic squeeze bottle with an aperture or openingdimensioned to aerosolize an aerosol formulation by forming a spray whensqueezed is used. The opening is usually found in the top of the bottle,and the top is generally tapered to partially fit in the nasal passagesfor efficient administration of the aerosol formulation. Preferably, thenasal inhaler will provide a metered amount of the aerosol formulation,for administration of a measured dose of the drug.

The compound, when it is desirable to deliver them systemically, may beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions.

Suitable lipophilic solvents or vehicles include fatty oils such assesame oil, or synthetic fatty acid esters, such as ethyl oleate ortriglycerides, or liposomes. Aqueous injection suspensions may containsubstances which increase the viscosity of the suspension, such assodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, thesuspension may also contain suitable stabilizers or agents whichincrease the solubility of the compounds to allow for the preparation ofhighly concentrated solutions.

Alternatively, the active compounds may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal or vaginal compositionssuch as suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, forexample, aqueous or saline solutions for inhalation, microencapsulated,encochleated, coated onto microscopic gold particles, contained inliposomes, nebulized, aerosols, pellets for implantation into the skin,or dried onto a sharp object to be scratched into the skin. Thepharmaceutical compositions also include granules, powders, tablets,coated tablets, (micro)capsules, suppositories, syrups, emulsions,suspensions, creams, drops or preparations with protracted release ofactive compounds, in whose preparation excipients and additives and/orauxiliaries such as disintegrants, binders, coating agents, swellingagents, lubricants, flavorings, sweeteners or solubilizers arecustomarily used as described above. The pharmaceutical compositions aresuitable for use in a variety of drug delivery systems. For a briefreview of methods for drug delivery, see Langer, Science 249: 1527-1533,1990, which is incorporated herein by reference.

The compounds of the invention may be administered per se (neat) or inthe form of a pharmaceutically acceptable salt. When used in medicinethe salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically acceptable salts thereof. Such salts include,but are not limited to, those prepared from the following acids:hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic,acetic, salicylic, p-toluene sulphonic, tartaric, citric, methanesulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, andbenzene sulphonic. Also, such salts can be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium or calcium salts of thecarboxylic acid group.

Suitable buffering agents include: acetic acid and a salt (1-2% w/v);citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v);and phosphoric acid and a salt (0.8-2% w/v). Suitable preservativesinclude benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9%w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

The pharmaceutical compositions of the invention contain an effectiveamount of a compound of the invention optionally included in apharmaceutically acceptable carrier. The term pharmaceuticallyacceptable carrier means one or more compatible solid or liquid filler,diluents or encapsulating substances which are suitable foradministration to a human or other vertebrate animal. The term carrierdenotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.The components of the pharmaceutical compositions also are capable ofbeing commingled with the compounds of the invention, and with eachother, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficiency.

The compounds of the invention may be delivered to the brain using aformulation capable of delivering a compound across the blood brainbarrier. One obstacle to delivering compounds to the brain is thephysiology and structure of the brain. The blood-brain barrier is madeup of specialized capillaries lined with a single layer of endothelialcells. The region between cells is sealed with a tight junction, so theonly access to the brain from the blood is through the endothelialcells. The barrier allows only certain substances, such as lipophilicmolecules through and keeps other harmful compounds and pathogens out.Thus, lipophilic carriers are useful for delivering non-lipohiliccompounds to the brain. For instance, DHA, a fatty acid naturallyoccurring in the human brain has been found to be useful for deliveringdrugs covalently attached thereto to the brain (Such as those describedin U.S. Pat. No. 6,407,137). U.S. Pat. No. 5,525,727 describes adihydropyridine pyridinium salt carrier redox system for the specificand sustained delivery of drug species to the brain. U.S. Pat. No.5,618,803 describes targeted drug delivery with phosphonate derivatives.U.S. Pat. No. 7,119,074 describes amphiphilic prodrugs of a therapeuticcompound conjugated to an PEG-oligomer/polymer for delivering thecompound across the blood brain barrier. The compounds described hereinmay be modified by covalent attachment to a lipophilic carrier orco-formulation with a lipophilic carrier. Others are known to those ofskill in the art.

The compounds of the invention may be delivered with other methods forenhancing memory retrieval or treating other symptoms or causes ofdisorders associated with the memory loss. For instance, environmentalenrichment (EE) has been used for enhancing memories. EE involvescreating a stimulating environment around a subject. Other therapeuticsmay also be combined to treat the underlying disorder or to enhancememory.

Combination Therapies

The invention includes combination therapies including the methods oftreating, alleviating, and/or preventing conditions described herein.Combination therapy includes administering one or more compounds of theinvention in combination with one or more pharmaceutically activeingredients or exposing the subject to cognitive behavioral therapy(CBT), psychotherapy, behavioral exposure treatments, virtual realityexposure (VRE) or cognitive remediation therapy.

In one aspect, the combination therapy is for a method of treating,alleviating, or preventing a neurological disorder. In one aspect, thecombination therapy is for methods of treating, alleviating, orpreventing Alzheimer's disease. The combination therapies comprise theadministration of an effective amount of one or more (e.g. one)compounds of the invention and the administration of an effective amountof one or more (e.g., one) other pharmaceutically active ingredients(e.g., drugs). The compounds of the invention and the otherpharmaceutically active ingredients can be administered separately(i.e., each is in its own separate dosage form), or the compounds of theinvention can be combined with the other pharmaceutically activeingredients in the same dosage form.

Pharmaceutically active ingredients that are useful in combinationtherapies of the invention include e.g., BACE inhibitors (beta secretaseinhibitors), muscarinic antagonists, cholinesterase inhibitors (e.g.,acetyl- and/or butyrylchlolinesterase inhibitors); gamma secretaseinhibitors; gamma secretase modulators; HMG-CoA reductase inhibitors;non-steroidal anti-inflammatory agents; N-methyl-D-aspartate receptorantagonists; anti-amyloid antibodies; vitamin E; nicotinic acetylcholinereceptor agonists; CB 1 receptor inverse agonists or CB 1 receptorantagonists; antibiotics; growth hormone secretagogues; histamine H3antagonists; AMPA agonists; PDE4 inhibitors; GABA inverse agonists;inhibitors of amyloid aggregation; glycogen synthase kinase betainhibitors; tau kinase inhibitors (e.g., GSK3beta inhibitors, cdk5inhibitors, ERK inhibitors), promoters of alpha secretase activity;PDE-10 inhibitors and cholesterol absorption inhibitors. Furtherexamples of pharmaceutically active ingredients that are useful forcombination therapies of the invention are(+)-2,3-dihydro-5,6-dimethoxy-2-[[1-(phenylmethyl)-4-piperidinyl]methy-1]-1H-inden-1-onehydrochloride, i.e., donepezil hydrochloride, available as the Aricept™brand of donepezil hydrochloride, Exelon (rivastigmine), Cognex(tacrine), anti-Abeta vaccine (active immunization), amyloid precursorprotein (APP) ligands, agents that upregulate insulin degrading enzymeand/or neprilysin, cholesterol lowering agents (for example, statinssuch as Atorvastatin, Fluvastatin, Lovastatin, Mevastatin, Pitavastatin,Pravastatin, Rosuvastatin, Simvastatin, and cholesterol absorptioninhibitor such as Ezetimibe, fibrates (for example, clofibrate,Clofibride, Etofibrate, Aluminium Clofibrate), LXR agonists, LRP mimics,5-HT6 receptor antagonists, nicotinic receptor agonists, H₃ receptorantagonists, other histone deacetylase inhibitors, hsp90 inhibitors,muscarinic receptor agonists, 5-HT6 receptor antagonists mGluR1 ormGluR5 positive allosteric modulators or agonists, mGluR2/3 antagonists,anti-inflammatory agents that can reduce neuroinflammation,prostaglandin EP2 receptor antagonists, PAI-1 inhibitors and agents thatcan induce Abeta efflux such as gelsolin.

Examples of combination therapies of the compounds of the invention withother pharmaceutically active ingredients include combinations with:anti-Alzheimer's agents, beta-secretase inhibitors, gamma-secretaseinhibitors, HMG-CoA reductase inhibitors, NSAID's including ibuprofen,N-methyl-D-aspartate (NMDA) receptor antagonists, such as memantine,cholinesterase inhibitors such as galantamine, rivastigmine, donepezil,and tacrine, vitamin E, CB-I receptor antagonists or CB-I receptorinverse agonists, antibiotics such as doxycycline and rifampin,anti-amyloid antibodies, or other pharmaceutically active ingredientsthat affect receptors or enzymes that either increase the efficacy,safety, convenience, or reduce unwanted side effects or toxicity of thecompounds of the invention. The compounds of the invention may also bedelivered in a cocktail of multiple HDAC inhibitors. Combinationtherapies of the invention may be in either unit dose or kit form.

The compounds of the invention are also useful in combination with knownpharmaceutically active ingredients such as anti-cancer agents.Combinations of the presently disclosed compounds with other anti-canceror chemotherapeutic agents are within the scope of the invention.Examples of such agents can be found in Cancer Principles and Practiceof Oncology by V. T. Devita and S. Hellman (editors), 6.sup.th edition(Feb. 15, 2001), Lippincott Williams & Wilkins Publishers. A person ofordinary skill in the art would be able to discern which combinations ofagents would be useful based on the particular characteristics of thedrugs and the cancer involved. Such anti-cancer agents include, but arenot limited to, the following: estrogen receptor modulators, androgenreceptor modulators, retinoid receptor modulators, cytotoxic/cytostaticagents, antiproliferative agents, prenyl-protein transferase inhibitors,HMG-CoA reductase inhibitors and other angiogenesis inhibitors,inhibitors of cell proliferation and survival signaling, apoptosisinducing agents, agents that interfere with cell cycle checkpoints,agents that interfere with receptor tyrosine kinases (RTKs) and cancervaccines. The compounds of the invention are particularly useful whenco-administered with radiation therapy.

In an embodiment, the instant compounds are also useful in combinationwith known anti-cancer agents including the following: estrogen receptormodulators, androgen receptor modulators, retinoid receptor modulators,cytotoxic agents, antiproliferative agents, prenyl-protein transferaseinhibitors, HMG-CoA reductase inhibitors, HIV protease inhibitors,reverse transcriptase inhibitors, and other angiogenesis inhibitors.

Additional combination therapies are discussed herein under theextinction learning section.

The invention also includes articles, which refers to any one orcollection of components. In some embodiments the articles are kits. Thearticles include pharmaceutical or diagnostic grade compounds of theinvention in one or more containers. The article may includeinstructions or labels promoting or describing the use of the compoundsof the invention. Compounds of the invention can be evaluated using avariety of methods known in the art. For example, the following methodscan be used to evaluate compounds of the invention: the inhibition ofHDAC activity can be determined using a trypsin-coupled protocol and/ora non-trypsin-coupled Caliper protocol (Schultz, B. E. Biochemistry,2004, 43, 11083 and U.S. patent application Ser. No. 61/628,562 entitled“Fluorescent Substrates for Determining Lysine Deacetylase Activity”filed Nov. 2, 2011); the inhibition of HDAC activity can be determinedin cells e.g., imaging of primary neuronal culture; learning tests suchas behavioral tests e.g., fear-conditioning can be performed asdescribed in Fischer et al., Neuron 48, 825-838 (2005); the ability ofthe compounds to reinstate learning behavior and to recover access tolong-term memories can be tested in CK-p25 Tg mice that have developedsynaptic and neuronal loss (Cruz, J., et al., Curr. Opin. Neurobiol. 14,390-394 (2004); Fisher, A. et al., Neuron 48, 471-83 (2003); Cruz, J. etal., Neuron 40, 471-83 (2003)); the effect of compounds on plasticityfactors in CK-p25 Tg mice with severe neurodegeneration can bedetermined by looking at hiippocampal neuronal loss, for example, bycomparing images showing hippocampal NeuN and MAP-2 staining and usingimmunoblots from the hippocampus and cortex of all groups; one or morespecific acetylation marks elicited by the compounds can be determinedwhich are relevant to the treatment of disease (e.g., Rubinstein Taybi);mass spectrometry can be used to identify changes in histone acetylationand methylation states induced in neurons by treatment with thecompounds; the identification of gene expression changes upon treatmentwith compounds of the invention in neurons can be determined using RNAfor transcript profile analysis on Illumina microarrays; and the abilityof the compounds to selectively inhibit HDACs1/2 to effect uniqueconformational changes in the enzymes induced by assembly into thesemulti-protein complexes can be evaluated by immunoprecipitating HDAC2complexes from mouse forebrain and by determining the presence andactivity of complex members (e.g., CoREST, mSin3a, and Mta3) using invitro assays.

The following Examples are illustrative and should not be interpreted inany way so as to limit the scope of the invention.

EXAMPLES Example 1 Preparation of Compounds of the Invention

Synthesis ofN-(2-amino-5-(thiophen-2-yl)phenyl)tetrahydro-2H-pyran-4-carboxamide(45)

To a solution of 4-bromo-2-nitroaniline (1, 10.0 g, 46.1 mmol, 1.0 eq.)in DMF (50 mL) at 0° C. was added sodium hydride (1.8 g, 73.7 mmol, 1.6eq.) slowly. After 30 minutes, a solution of di-tert-butyl dicarbonate(12.1 g, 55.3 mmol, 1.2 eq.) in DMF (50 mL) was added dropwise. Thereaction mixture was then stirred for 16 h at room temperature. Thereaction was quenched with water. The product was extracted with methyltertiary butyl ether. The organic layer was washed with water and brine.The combined organic layers were dried over anhydrous sodium sulfate,filtered and concentrated in vacuo. The product was purified by columnchromatography (silica gel, 0-20% EtOAc/hexanes) to give tert-butyl(4-bromo-2-nitrophenyl)carbamate (7.5 g, 51% yield) as yellow solid.

A mixture of tert-butyl (4-bromo-2-nitrophenyl)carbamate (6.0 g, 18.92mmol, 1.0 eq.), thiophen-2-ylboronic acid (3.2 g, 24.6 mmol, 1.3 eq.),potassium carbonate (7.84 g, 56.8 mmol, 3.0 eq.) andtetrakis(triphenylphosphine)palladium(0) (1.53 g, 1.32 mmol, 0.07 eq.),tri-tolylphosphine in DME/H2O (105 mL) was first degassed then heated to90° C. for 20 h. The reaction was then filtered through Celite. Theproduct was extracted with ethyl acetate. The combined organic layerswere washed with water and brine, dried over anhydrous Na₂SO₄, filteredand concentrated under reduced pressure. The crude product was purifiedby flash column chromatography (silica gel, 2% EtOAc/hexanes) to obtainpure tert-butyl (2-nitro-4-(thiophen-2-yl)phenyl)carbamate (4.42 g, 73%yield).

To a solution of tert-butyl (2-nitro-4-(thiophen-2-yl)phenyl)carbamate(2 g, 6.2 mmol, 1.0 eq.) in ethanol (20 mL) and methanol (20 mL) wasadded 10% Pd/C (0.66 g, 0.1 eq.). The reaction mixture was stirred 12 hunder a hydrogen atmosphere. The reaction was filtered and the filtratewas concentrated under reduced pressure to give tert-butyl(2-amino-4-(thiophen-2-yl)phenyl)carbamate (1.25 g, 69% yield) as anoff-white solid.

To a solution of tert-butyl (2-nitro-4-(thiophen-2-yl)phenyl)carbamate(1.6 g, 4.99 mmol, 1 eq.) in methanol (20 mL) was added hydrazinehydrate (14 mL) and ferric chloride (0.05 g, 0.3 mmol, 0.06 eq.). Theresulting mixture was warmed to 60° C. and stirred for 2 h. The reactionwas then filtered through Celite, the solids were washed with MeOH. Thefiltrate was concentrated under reduced pressure. Water was added to theresidue and the suspension was stirred for 1 h. The obtained solid wasfiltered, washed with hexanes then dried to yield tert-butyl(2-amino-4-(thiophen-2-yl)phenyl)carbamate (1.2 g, 83% yield).

A solution of tert-butyl (2-amino-4-(thiophen-2-yl)phenyl)carbamate (1.2g, 4.13 mmol, 1.0 eq.), tetrahydro-2H-pyran-4-carboxylic acid (0.65 g,4.96 mmol, 1.2 eq.), HATU (3.14 g, 8.27 mmol, 2.0 eq.) and Hünigs base(1.80 mL, 10.33 mmol, 2.5 eq.) in DMF (15 mL) was stirred for 18 h atroom temperature. The reaction mixture was diluted with water. The solidwas isolated by filtration and washed with hexanes to afford tert-butyl(2-(tetrahydro-2H-pyran-4-carboxamido)-4-(thiophen-2-yl)phenyl)carbamate(1.3 g, 78% yield).

To a stirred solution of tert-butyl(2-(tetrahydro-2H-pyran-4-carboxamido)-4-(thiophen-2-yl)phenyl)carbamatein dichloromethane (20 mL) was added trifluoroacetic acid (7 mL) at roomtemperature. The reaction mixture was stirred at room temperature for 2h. the solvents were then removed under reduced pressure. A saturatedaqueous solution of sodium bicarbonate was added. The product wasextracted with EtOAc, washed with water and brine, dried, filtered andconcentrated under reduced pressure. The crude product was purified bycolumn chromatography (silica gel, MeOH/CH₂Cl₂) to affordN-(2-amino-5-(thiophen-2-yl)phenyl)tetrahydro-2H-pyran-4-carboxamide asa beige solid (1.1 g, 86% yield). ESI+MS: m/z 303 ([M+H]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 9.12 (s, 1H), 7.51 (d, J=2.0 Hz, 1H), 7.34 (d, J=5.0Hz, 1H), 7.24-7.18 (m, 2H), 7.03 (dd, J=5.5; 3.5 Hz, 1H), 6.75 (d, J=8.5Hz, 1H), 5.05 (s, 1H), 3.91 (dd, J=11.0; 2.0 Hz, 2H), 3.36 (dt, J=11.0;2.0 Hz, 2H), 2.68-2.62 (m, 1H), 1.80-1.64 (m, 4H).

One skilled in the art will recognize that other compounds describedbelow were prepared in a similar manner to the procedures describedabove.

N-(2-amino-5-(thiophen-2-yl)phenyl)cyclohexanecarboxamide (30) wasprepared by substituting tetrahydro-2H-pyran-4-carboxylic acid in Scheme5 with cyclohexane carboxylic acid. ESI+MS: m/z 301 ([M+H]⁺), 1H NMR(500 MHz, d⁶-DMSO): δ 9.06 (s, 1H), 7.51 (d, J=1.0 Hz, 1H), 7.34 (d,J=5.5 Hz, 1H), 7.26-7.14 (m, 2H), 7.04 (t, J=7.04 Hz, 1H), 6.75 (d,J=8.5 Hz, 1H), 5.06 (s, 2H), 2.39 (t, J=11.5 Hz, 1H), 1.84 (d, J=12.0Hz, 2H), 1.76 (d, J=12.0 Hz, 2H), 1.66 (d, J=12.0 Hz, 1H), 1.43 (q,J=11.5 Hz, 2H), 1.10-1.35 (m, 3H).

N-(2-amino-5-(thiophen-2-yl)phenyl)-2-(tetrahydro-2H-pyran-4-yl)acetamide(8) was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acidin Scheme 5 with 2-(tetrahydro-2H-pyran-4-yl)acetic acid. ESI+MS: m/z316 ([M]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 9.21 (s, 1H), 7.50 (d, J=2.0Hz, 1H), 7.26-7.18 (m, 2H), 7.04 (dd, J=4.0, 5.0 Hz, 1H), 6.76 (d, J=8.0Hz, 1H), 5.20 (bs, 2H), 3.84 (dd, J=11.0, 3.0 Hz, 2H), 3.81 (t, J=11.0Hz, 2H), 2.29 (d, J=7.0 Hz, 2H), 2.05-1.95 (m, 1H), 1.63 (d, J=11.0 Hz,2H), 1.35-1.20 (m, 2H).

1-acetyl-N-(2-amino-5-(thiophen-2-yl)phenyl)piperidine-4-carboxamide(35) was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acidin Scheme 5 with 1-acetylpiperidine-4-carboxylic acid. ESI+MS: m/z 316([M]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 9.19 (s, 1H), 7.50 (d, J=1.5 Hz,1H), 7.34 (d, J=4.5 Hz, 1H), 7.24-7.18 (m, 2H), 7.04 (dd, J=4.0, 5.5 Hz,1H), 6.75 (d, J=8.5 Hz, 1H), 5.06 (s, 2H), 4.35 (d, J=13.0 Hz, 1H), 3.79(d, J=13.5 Hz, 2H), 3.01 (t, J=11.5 Hz, 1H), 2.60-2.50 (m, 1H), 2.29 (d,J=7.0 Hz, 2H), 2.05-1.95 (m, 4H), 1.80-1.65 (m, 2H), 1.25-1.10 (m, 1H),1.10-1.00 (m, 1H).

1-acetyl-N-(4-amino-2′,3′,4′,5′-tetrahydro-[1,1′-biphenyl]-3-yl)piperidine-4-carboxamide(37) was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acidin Scheme 5 with 1-acetylpiperidine-4-carboxylic acid and bysubstituting thiophen-2-ylboronic acid with cyclohex-1-en-1-ylboronicacid in Scheme 2. ESI+MS: m/z 342 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ9.08 (s, 1H), 7.22 (d, J=2.0 Hz, 1H), 6.97 (dd, J=2.0, 8.0 Hz, 1H), 6.66(d, J=8.0 Hz, 1H), 5.91 (bs, 1H), 4.80 (s, 2H), 4.39 (d, J=13.0 Hz, 1H),3.86 (d, J=14.0 Hz, 1H), 3.06 (t, J=12.5 Hz, 1H), 2.66-2.54 (m, 2H),2.33-2.30 (m, 2H), 2.16-2.10 (m, 2H), 2.01 (s, 3H), 1.86-1.78 (m, 2H),1.72-1.66 (m, 2H), 1.64-1.54 (m, 3H), 1.50-1.40 (m, 1H).

N-(4-amino-2′,3′,4′,5′-tetrahydro-[1,1′-biphenyl]-3-yl)tetrahydro-2H-pyran-4-carboxamide(59) was prepared by substituting thiophen-2-ylboronic acid in Scheme 2with cyclohex-1-en-1-ylboronic acid. ESI+MS: m/z 301 ([M+H]⁺).

1-acetyl-N-(2-amino-5-(cyclopent-1-en-1-yl)phenyl)piperidine-4-carboxamide(38) was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acidin Scheme 5 with 1-acetylpiperidine-4-carboxylic acid and bysubstituting thiophen-2-ylboronic acid in Scheme 2 withcyclopent-1-en-1-ylboronic acid. ESI+MS: m/z 328 ([M+H]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 9.12 (s, 1H), 7.24 (s, 1H), 7.05 (d, J=8.0 Hz, 1H),6.67 (d, J=8.0 Hz, 1H), 5.92 (bs, 1H), 4.88 (s, 2H), 4.39 (d, J=13.0 Hz,1H), 4.12-4.05 (m, 1H), 3.86 (d, J=14.0 Hz, 1H), 3.06 (t, J=12.5 Hz,1H), 2.65-2.40 (m, 5H), 2.01 (s, 3H), 1.95-1.78 (m, 4H), 1.65-1.52 (m,1H), 1.50-1.38 (m, 1H).

N-(2-amino-5-(thiophen-2-yl)phenyl)-1-methylpiperidine-4-carboxamide(34) was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acidin Scheme 5 with 1-methylpiperidine-4-carboxylic acid. ESI+MS: m/z 316([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 9.12 (s, 1H), 7.51 (s, 1H), 7.34(d, J=5.0 Hz, 1H), 7.24-7.18 (m, 2H), 7.04 (t, J=4.0 Hz, 1H), 6.75 (t,J=8.5 Hz, 1H), 5.06 (bs, 2H), 2.82 (d, J=11.0 Hz, 2H), 2.35-2.25 (m,1H), 1.80-1.60 (m, 6H).

2-(1-acetylpiperidin-4-yl)-N-(2-amino-5-(thiophen-2-yl)phenyl)acetamide(10) was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acidin Scheme 5 with 2-(1-acetylpiperidin-4-yl)acetic acid. ESI+MS: m/z 316([M]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 9.19 (s, 1H), 7.50 (d, J=1.5 Hz,1H), 7.34 (d, J=4.5 Hz, 1H), 7.24-7.18 (m, 2H), 7.04 (dd, J=4.0, 5.5 Hz,1H), 6.75 (d, J=8.5 Hz, 1H), 5.06 (s, 2H), 4.35 (d, J=13.0 Hz, 1H), 3.79(d, J=13.5 Hz, 2H), 3.01 (t, J=11.5 Hz, 1H), 2.60-2.50 (m, 1H), 2.29 (d,J=7.0 Hz, 2H), 2.05-1.95 (m, 4H), 1.80-1.65 (m, 2H), 1.25-1.10 (m, 1H),1.10-1.00 (m, 1H).

N-(2-amino-5-(5-chlorothiophen-2-yl)phenyl)tetrahydro-2H-pyran-4-carboxamide(48) was prepared by substituting thiophen-2-ylboronic acid in Scheme 2with (5-chlorothiophen-2-yl)boronic acid. ESI+MS: m/z 337 ([M+H]⁺), 1HNMR (300 MHz, d⁶-DMSO): δ 9.15 (s, 1H), 7.46 (d, J=3.0 Hz, 1H), 7.18(dd, J=3.0, 9.0 Hz, 1H), 7.09-7.03 (m, 2H), 6.76 (d, J=9.0 Hz, 1H), 5.17(bs, 2H), 3.88-3.82 (m, 2H), 3.42-3.24 (m, 2H), 2.66-2.46 (m, 1H),1.80-1.60 (m, 4H).

N-(4-amino-[1,1′-biphenyl]-3-yl)tetrahydro-2H-pyran-4-carboxamide (49)was prepared by substituting thiophen-2-ylboronic acid in Scheme 2 withphenylboronic acid. ESI+MS: m/z 296.9 ([M+H]⁺).

N-(2-amino-5-(thiophen-2-yl)phenyl)cyclopent-1-enecarboxamide (17) wasprepared by substituting tetrahydro-2H-pyran-4-carboxylic acid in Scheme5 with cyclopent-1-enecarboxylic acid. ESI+MS: m/z 285 ([M+H]⁺), 1H NMR(500 MHz, d⁶-DMSO): δ 9.12 (s, 1H), 7.40 (d, J=2 Hz, 1H), 7.35 (dd, J=5,1 Hz, 1H), 7.26 (dd, J=8; 2 Hz, 1H), 7.22 (dd, J=4 Hz; 1.5 Hz, 1H), 7.04(dd, J=5; 4.5 Hz, 1H), 6.78 (d, J=8.5 Hz, 1H), 6.73-6.68 (m, 1H), 5.06(s, 2H), 2.61-2.57 (m, 2H), 2.51-2.48 (m, 2H), 1.93-1.90 (m, 2H).

N-(2-amino-5-cyclopentylphenyl)cyclohex-1-enecarboxamide (31) wasprepared by substituting tetrahydro-2H-pyran-4-carboxylic acid in Scheme5 with cyclohex-1-enecarboxylic acid and by substitutingthiophen-2-ylboronic acid in Scheme 2 with cyclopentylboronic acid.ESI+MS: m/z 285 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 8.94 (s, 1H),6.98 (d, J=1.5 Hz, 1H), 6.81 (dd, J=1.5, 8.5 Hz, 1H), 6.70-6.64 (m, 2H),4.56 (s, 2H), 2.85-2.75 (m, 1H), 2.30-2.24 (m, 2H), 2.20-2.12 (m, 2H),1.96-1.88 (m, 2H), 1.76-1.66 (m, 2H), 1.66-1.52 (m, 6H), 1.48-1.38 (m,2H).

N-(2-amino-5-(thiophen-2-yl)phenyl)cyclopentanecarboxamide (16) wasprepared by substituting tetrahydro-2H-pyran-4-carboxylic acid in Scheme5 with cyclopentanecarboxylic acid. ESI+MS: m/z 287 ([M+H]⁺), 1H NMR(500 MHz, d⁶-DMSO): δ 9.15 (s, 1H), 7.52 (s, 1H), 7.35 (d, J=4 Hz, 1H),7.18-7.26 (m, 2H), 7.04 (t, J=4 Hz, 1H), 6.75 (d, J=8.5 Hz, 1H), 5.06(s, 2H), 2.84 (q, J=7.5 Hz, 1H), 1.95-1.65 (m, 2H), 1.65-1.30 (m, 4H),1.30-1.50 (m, 2H).

N-(2-amino-5-(thiophen-2-yl)phenyl)cyclobutanecarboxamide (13) wasprepared by substituting tetrahydro-2H-pyran-4-carboxylic acid in Scheme5 with cyclobutanecarboxylic acid. ESI+MS: m/z 273 ([M+H]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 9.01 (s, 1H), 7.52 (s, 1H), 7.35 (d, J=4.0 Hz, 1H),7.18-7.25 (m, 2H), 7.04 (t, J=5.0 Hz, 1H), 6.75 (d, J=8.5 Hz, 1H), 5.06(s, 2H), 3.27 (q, J=8.0 Hz, 1H), 2.25 (q, J=9.0 Hz, 2H), 2.30-2.15 (m,2H), 2.10-1.80 (m, 1H), 1.80-1.55 (m, 1H).

N-(2-amino-5-(thiophen-2-yl)phenyl)cyclopropanecarboxamide (12) wasprepared by substituting tetrahydro-2H-pyran-4-carboxylic acid in Scheme5 with cyclopropanecarboxylic acid. ESI+MS: m/z 259 ([M+H]⁺), 1H NMR(500 MHz, d⁶-DMSO): δ 9.46 (s, 1H), 7.56 (s, 1H), 7.34 (d, J=4.5 Hz,1H), 7.25-7.16 (m, 2H), 7.04 (t, J=3.5 Hz, 1H), 6.75 (d, J=9 Hz, 1H),5.09 (s, 2H), 1.84 (d, J=4.5 Hz, 1H), 0.80 (t, J=3 Hz, 4H).

N-(2-amino-5-(thiophen-2-yl)phenyl)-2-cyclopropylacetamide (11) wasprepared by substituting tetrahydro-2H-pyran-4-carboxylic acid in Scheme5 with 2-cyclopropylacetic acid. ESI+MS: m/z 273 ([M+H]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 8.84 (s, 1H), 7.30 (d, J=2.5 Hz, 1H), 7.12 (dd, J=1.0,6.0 Hz, 1H), 7.25-7.19 (m, 2H), 6.81 (dd, J=4.5, 6.5 Hz, 1H), 6.52 (d,J=10.5 Hz, 1H), 4.86 (bs, 2H), 2.02 (d, J=9.0 Hz, 2H), 1.13-1.03 (m,1H), 0.52-0.46 (m, 2H), 0.25-0.18 (m, 2H).

N-(4-amino-2′,3′,4′,5′-tetrahydro-[1,1′-biphenyl]-3-yl)tetrahydro-2H-pyran-4-carboxamide(50) was prepared by substituting thiophen-2-ylboronic acid in Scheme 2with cyclohex-1-en-1-ylboronic acid. ESI+MS: m/z 301 ([M+H]⁺), 1H NMR(500 MHz, d⁶-DMSO): δ 9.04 (s, 1H), 7.23 (d, J=2.0 Hz, 1H), 6.97 (dd,J=2.0, 8.0 Hz, 1H), 6.65 (d, J=8.0 Hz, 1H), 5.91 (bs, 1H), 4.80 (s, 2H),3.94-3.86 (m, 2H), 3.40-3.30 (m, 2H), 2.66-2.56 (m, 1H), 2.30-2.22 (m,2H), 2.15-2.09 (m, 2H), 1.75-1.60 (m, 6H), 1.60-1.53 (m, 2H).

N-(2-amino-5-(thiophen-2-yl)phenyl)-1-methyl-2-oxopiperidine-4-carboxamide(40) was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acidin Scheme 5 with 1-methyl-2-oxopiperidine-4-carboxylic acid. ESI+MS: m/z330 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 9.22 (s, 1H), 7.49 (s, 1H),7.34 (d, J=5.0 Hz, 1H), 7.26-7.18 (m, 2H), 7.04 (t, J=5.0 Hz, 1H), 6.75(d, J=8.5 Hz, 1H), 5.08 (s, 2H), 2.98-2.90 (m, 1H), 2.50-2.35 (m, 3H),2.13-2.05 (m, 1H), 1.92-1.82 (m, 1H).

N-(4-amino-2′,3′,4′,5′-tetrahydro-[1,1′-biphenyl]-3-yl)-1-methyl-2-oxopiperidine-4-carboxamide(41) was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acidin Scheme 5 with 1-methyl-2-oxopiperidine-4-carboxylic acid and bysubstituting thiophen-2-ylboronic acid in Scheme 2 withcyclohex-1-en-1-ylboronic acid. ESI+MS: m/z 328 ([M+H]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 9.14 (s, 1H), 7.23 (s, 1H), 6.98 (d, J=6.5 Hz, 1H),6.66 (d, J=6.5 Hz, 1H), 5.91 (bs, 1H), 4.83 (s, 2H), 3.34-3.26 (m, 2H),2.96-2.86 (m, 1H), 2.82 (s, 3H), 2.44-2.32 (m, 2H), 2.30-2.22 (m, 2H),2.16-2.02 (m, 3H), 1.92-1.80 (m, 1H), 1.72-1.64 (m, 2H), 1.60-1.52 (m,2H).

1-acetyl-N-(2-amino-5-(thiophen-2-yl)phenyl)azetidine-3-carboxamide (42)was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acid inScheme 5 with 1-acetylazetidine-3-carboxylic acid. ESI+MS: m/z 316([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 9.29 (s, 1H), 7.51 (d, J=2.0 Hz,1H), 7.35 (d, J=4.5 Hz, 1H), 7.24 (d, J=2.5, 8.5 Hz, 1H), 7.21 (d, J=3.0Hz, 1H), 7.04 (dd, J=3.5, 5.0 Hz, 1H), 6.75 (d, J=8.5 Hz, 1H), 5.15 (bs,2H), 4.30-4.20 (m, 2H), 4.06-3.90 (m, 2H), 3.60-3.50 (m, 1H), 1.78 (s,3H).

N-(2-amino-5-(thiophen-2-yl)phenyl)azetidine-3-carboxamide (43) wasprepared by substituting tetrahydro-2H-pyran-4-carboxylic acid in Scheme5 with 1-(tert-butoxycarbonyl)azetidine-3-carboxylic acid. ESI+MS: m/z274 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 9.30 (s, 1H), 7.54 (s, 1H),7.34 (d, J=5.0 Hz, 1H), 7.24-7.18 (m, 2H), 7.05-7.02 (m, 1H), 6.74 (d,J=8.5 Hz, 1H), 5.13 (s, 2H), 3.80-3.60 (m, 4H).

2-(1-acetylazetidin-3-yl)-N-(2-amino-5-(thiophen-2-yl)phenyl)acetamide(9) was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acidin Scheme 5 with 2-(1-acetylazetidin-3-yl)acetic acid. ESI+MS: m/z 330([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 9.19 (s, 1H), 7.49 (d, J=2.0 Hz,1H), 7.34 (d, J=5.0 Hz, 1H), 7.22 (dd, J=2.0, 8.0 Hz, 1H), 7.19 (d,J=3.0 Hz, 1H), 7.03 (t, J=5.0 Hz, 1H), 6.74 (d, J=8.0 Hz, 1H), 5.09 (s,2H), 4.24 (t, J=8.5 Hz, 1H), 3.95 (t, J=9.5 Hz, 1H), 3.86 (dd, J=6.0,8.5 Hz, 1H), 3.57 (dd, J=6.0, 9.5 Hz, 1H), 2.96-2.86 (m, 1H), 2.70 (d,J=7.5 Hz, 2H), 1.74 (s, 3H).

N-(2-amino-5-(pyridin-3-yl)phenyl)tetrahydro-2H-pyran-4-carboxamide (51)was prepared by substituting thiophen-2-ylboronic acid in Scheme 2 withpyridin-3-ylboronic acid. ESI+MS: m/z 298 ([M+H]⁺), 1H NMR (500 MHz,d⁶-DMSO): δ 9.15 (s, 1H), δ 8.75 (s, 1H), δ 8.44 (d, J=4.0 Hz, 1H), δ7.89 (d, J=8.5 Hz, 1H), δ 7.59 (d, J=1.5, 1H), δ 7.39 (dd, J=7.5; 4.5Hz, 1H), δ 7.31 (dd, J=8.0; 1.5 Hz, 1H), δ 6.84 (d, J=8.5 Hz, 1H), δ5.11 (s, 2H), δ 3.92 (d, J=9.5 Hz, 2H), δ 3.37 (t, J=9.5 Hz, 2H), δ2.70-2.61 (m, 1H), δ 1.80-1.63 (m, 4H)

N-(4-amino-4′-chloro-[1,1′-biphenyl]-3-yl)tetrahydro-2H-pyran-4-carboxamide(53) was prepared by substituting thiophen-2-ylboronic acid in Scheme 2with (4-chlorophenyl)boronic acid. ESI+MS: m/z 331 ([M+H]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 9.12 (s, 1H), 7.55 (s, 1H), 7.53 (d, J=9.0 Hz, 2H),7.42 (d, J=9.0 Hz, 2H), 7.25 (dd, J=8.5 Hz; 1 Hz, 1H), 6.80 (d, J=8.0Hz, 1H), 5.07 (s, 2H), 3.91 (d, J=10.5 Hz, 2H), 3.40-3.31 (m, 2H),2.70-2.62 (m, 1H), 1.77-1.63 (m, 4H).

N-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)tetrahydro-2H-pyran-4-carboxamide(54) was prepared by substituting thiophen-2-ylboronic acid in Scheme 2with (4-fluorophenyl)boronic acid. ESI+MS: m/z 331 ([M+H]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 9.12 (s, 1H), 7.55 (s, 1H), 7.53 (d, J=9.0 Hz, 2H),7.42 (d, J=9.0 Hz, 2H), 7.25 (dd, J=8.5 Hz; 1 Hz, 1H), 6.80 (d, J=8.0Hz, 1H), 5.07 (s, 2H), 3.91 (d, J=10.5 Hz, 2H), 3.40-3.31 (m, 2H),2.70-2.62 (m, 1H), 1.77-1.63 (m, 4H).

N-(2-amino-5-(cyclopent-1-en-1-yl)phenyl)tetrahydro-2H-pyran-4-carboxamide(55) was prepared by substituting thiophen-2-ylboronic acid in Scheme 2with cyclopent-1-en-1-ylboronic acid. ESI+MS: m/z 287 ([M+H]⁺), 1H NMR(500 MHz, d⁶-DMSO): δ 9.09 (s, 1H), 7.25 (d, J=2.0 Hz, 1H), 7.05 (dd,J=2.0, 9.0 Hz, 1H), 6.68 (d, J=8.0 Hz, 1H), 5.93 (s, 1H), 4.92 (bs, 2H),3.94-3.86 (m, 2H), 3.42-3.20 (m, 2H), 2.66-2.58 (m, 1H), 2.58-2.50 (m,2H), 2.46-2.40 (m, 2H), 1.91 (quintet, J=7.5 Hz, 2H), 1.80-1.60 (m, 4H)

N-(2-amino-5-(thiophen-2-yl)phenyl)tetrahydrofuran-3-carboxamide (44)was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acid inScheme 5 with tetrahydrofuran-3-carboxylic acid. ESI+MS: m/z 289([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 9.27 (s, 1H), 7.50 (s, 1H), 7.35(d, J=5.0 Hz, 1H), 7.25-7.18 (m, 2H), 7.04 (t, J=4.5 Hz, 1H), 6.76 (d,J=8.0 Hz, 1H), 5.11 (s, 2H), 3.96 (t, J=8 Hz, 1H), 3.82-3.68 (m, 3H),3.25-3.15 (m, 1H), 2.10 (q, J=7.5 Hz, 2H)

1-acetyl-N-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)piperidine-4-carboxamide(39) was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acidin Scheme 5 with 1-acetylpiperidine-4-carboxylic acid and bysubstituting thiophen-2-ylboronic acid in Scheme 2 with(4-fluorophenyl)boronic acid. ESI+MS: m/z 356 ([M+H]⁺), 1H NMR (500 MHz,d⁶-DMSO): δ 9.16 (s, 1H), 7.58-7.46 (m, 3H), 7.28-7.16 (m, 3H), 6.80 (d,J=8 Hz, 1H), 5.00 (s, 2H), 4.40 (d, J=12.5 Hz, 1H), 3.87 (d, J=13.5 Hz,1H), 3.08 (t, J=12.5 Hz, 1H), 2.70-2.55 (m, 2H), 2.01 (s, 3H), 1.86 (t,J=13.5 Hz, 2H), 1.68-1.55 (m, 1H), 1.55-1.40 (m, 1H)

N-(2-amino-5-(pyridin-4-yl)phenyl)tetrahydro-2H-pyran-4-carboxamide (52)was prepared by substituting thiophen-2-ylboronic acid in Scheme 2 withpyridin-4-ylboronic acid. ESI+MS: m/z 298 ([M+H]⁺), 1H NMR (500 MHz,d⁶-DMSO): δ 9.13 (s, 1H), 8.50 (d, J=5 Hz, 2H), 7.71 (d, J=1.5 Hz, 1H),7.53 (d, J=5.5 Hz, 2H), 7.42 (dd, J=8.5; 2 Hz, 1H), 6.83 (d, J=8.5 Hz,1H), 5.27 (s, 2H), 3.92 (d, J=9 Hz, 2H), 3.37 (dt, J=10.5 Hz; 1.5 Hz,2H), 2.70-2.62 (m, 1H), 1.80-1.64 (m 4H)

N-(2-amino-5-(thiophen-2-yl)phenyl)cyclohex-1-enecarboxamide (32) wasprepared by substituting tetrahydro-2H-pyran-4-carboxylic acid in Scheme5 with cyclohex-1-enecarboxylic acid. ESI+MS: m/z 299 ([M+H]⁺), 1H NMR(500 MHz, d⁶-DMSO): δ 9.01 (s, 1H), 7.40 (s, 1H), 7.34 (d, J=4.5 Hz,1H), 7.28-7.18 (m, 2H), 7.04 (t, J=4.0 Hz, 1H), 6.77 (d, J=8.0 Hz, 1H),6.72 (bs, 1H), 5.01 (s, 2H), 2.32-2.26 (m, 2H), 2.22-2.14 (m, 2H),1.66-1.55 (m, 4H).

N-(4-amino-2′,3′,4′,5′-tetrahydro-[1,1′-biphenyl]-3-yl)cyclohex-1-enecarboxamide(33) was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acidin Scheme 5 with cyclohex-1-enecarboxylic acid and by substitutingthiophen-2-ylboronic acid in Scheme 2 with cyclohex-1-en-1-ylboronicacid.

ESI+MS: m/z 297 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 8.95 (s, 1H),7.16 (s, 1H), 7.01 (d, J=8.0 Hz, 1H), 6.73-6.66 (m, 2H), 5.93 (s, 1H),4.92 (bs, 2H), 2.30-2.22 (m, 4H), 2.20-2.10 (m, 4H), 1.72-1.54 (m, 8H).

N-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)cyclopent-1-enecarboxamide(18) was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acidin Scheme 5 with cyclopent-1-enecarboxylic acid and by substitutingthiophen-2-ylboronic acid in Scheme 2 with (4-fluorophenyl)boronic acid.

ESI+MS: m/z 297 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 9.09 (s, 1H),7.54 (dd, J=8.5; 5.5 Hz, 2H), 7.41 (d, J=2 Hz, 1H), 7.25 (dd, J=8; 2 Hz,1H), 7.19 (t, J=12.5 Hz, 2H), 6.82 (d, J=8 Hz, 1H), 6.69 (s, 1H), 4.98(s, 2H), 2.62-2.54 (m, 2H), 2.54-2.43 (m, 2H), 1.91 (q, J=7.5 Hz, 2H).

N-(2-amino-5-(thiophen-2-yl)phenyl)-3-oxocyclobutanecarboxamide (15) wasprepared by substituting tetrahydro-2H-pyran-4-carboxylic acid in Scheme5 with 3-oxocyclobutanecarboxylic acid. ESI+MS: m/z 287 ([M+H]⁺), 1H NMR(500 MHz, d⁶-DMSO): δ 9.44 (s, 1H), 7.54 (d, J=2.0 Hz, 1H), 7.34 (d,J=4.0 Hz, 1H), 7.25-7.20 (m, 2H), 7.04 (dd, J=4.0, 5.5 Hz, 1H), 6.76 (d,J=8.5 Hz, 1H), 5.13 (s, 2H), 3.43-3.35 (m, 1H), 3.32-3.27 (m, 4H).

N-(2-amino-5-(thiophen-2-yl)phen-2-phenyl)tetrahydro-2H-pyran-2-carboxamide(56) was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acidin Scheme 5 with tetrahydro-2H-pyran-2-carboxylic acid. ESI+MS: m/z 303([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 8.97 (s, 1H), 7.51 (s, 1H), 7.35(d, J=4.5 Hz, 1H), 7.24-7.20 (m, 2H), 7.04 (t, J=4.5 Hz, 1H), 6.78 (d,J=8.5 Hz, 1H), 4.97 (s, 2H), 4.04-3.94 (dd, J=11.5 Hz, 2H), 3.54-3.51(m, 1H), 1.95-1.84 (m, 2H), 1.54-1.49 (m, 4H).

N-(2-amino-5-(thiophen-2-yl)phenyl)-3-(piperidin-4-yl)propanamide (158)was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acid inScheme 5 with 3-(1-(tert-butoxycarbonyl)piperidin-4-yl)propanoic acid.ESI+MS: m/z 330 ([M+H]⁺). 1H NMR (500 MHz, d⁶-DMSO): δ 9.14 (s, 1H),7.49 (s, 1H), 7.34 (d, J=5.0 Hz, 1H), 7.22-7.18 (m, 2H), 7.04-7.03 (m,1H), 6.74 (d, J=8.5 Hz, 1H), 5.06 (s, 2H), 3.06 (d, J=12.0 Hz, 2H), 2.60(t, J=11.0 Hz, 2H), 2.36 (t, J=8.0 Hz, 2H), 1.71 (d, J=12.0 Hz, 2H),1.57-1.52 (m, 2H), 1.46-1.42 (m, 1H), 1.16-1.11 (m, 4H).

N-(2-amino-5-(pyridin-4-yl)phenyl)cyclohexanecarboxamide (163) wasprepared by substituting tetrahydro-2H-pyran-4-carboxylic acid in Scheme5 with cyclohexane carboxylic acid and by substitutingthiophen-2-ylboronic acid in Scheme 2 with pyridin-4-ylboronic acid.ESI+MS: m/z 296 ([M]⁺); 1H NMR (500 MHz, d⁶-DMSO): δ 9.07 (s, 1H), 8.50(d, J=5.5 Hz, 2H), 7.71 (s, 1H), 7.53 (d, J=5.5 Hz, 2H), 7.41-7.39 (m,1H), 6.82 (d, J=8.5 Hz, 1H), 5.24 (s, 2H), 2.40 (t, J=12.0 Hz, 1H),1.87-1.64 (m, 5H), 1.46-1.39 (m, 2H), 1.32-1.18 (m, 3H).

N-(2-amino-5-(thiophen-2-yl)phenyl)tetrahydrofuran-2-carboxamide (171)was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acid inScheme 5 with tetrahydrofuran-2-carboxylic acid. ESI+MS: m/z 289 ([M]⁺);1H NMR (500 MHz, d⁶-DMSO): δ 9.15 (s, 1H), 7.50 (s, 1H), 7.35 (d, J=5.0Hz, 1H), 7.25-7.22 (m, 2H), 7.04 (t, J=4.5 Hz, 1H), 6.75 (d, J=8.5 Hz,1H), 5.00 (s, 2H), 4.44-4.41 (m, 1H), 4.01 (q, J=7.0 Hz, 1H), 3.83 (q,J=7.0 Hz, 1H), 2.22-2.18 (m, 1H), 2.03-1.98 (m, 1H), 1.92-1.85 (m, 2H).

N-(2-amino-5-(thiophen-2-yl)phenyl)piperidine-2-carboxamide (173) wasprepared by substituting tetrahydro-2H-pyran-4-carboxylic acid in Scheme5 with 1-(tert-butoxycarbonyl)piperidine-2-carboxylic acid. ESI+MS: m/z302 ([M]⁺); 1H NMR (500 MHz, d⁶-DMSO): δ 8.86 (bs, 1H), 7.55 (s, 1H),7.35 (d, J=4.5 Hz, 1H), 7.23-7.19 (m, 2H), 7.05-7.03 (m, 1H), 6.76 (d,J=8.0 Hz, 1H), 5.05 (s, 2H), 3.23 (d, J=6.5 Hz, 1H), 2.99 (d, J=11.5 Hz,1H), 2.57 (t, J 11.0 Hz, 1H), 1.85-1.78 (m, 2H), 1.52-1.34 (m, 4H).

N-(2-amino-5-(thiophen-2-yl)phenyl)-2-hydroxy-2-methylpropanamide (200)was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acid inScheme 5 with 2-hydroxy-2-methylpropanoic acid. ESI+MS: m/z 277([M+H]⁺); 1H NMR (500 MHz, d⁶-DMSO): δ 9.04 (s, 1H), 7.61 (s, 1H), 7.35(d, J=4.5 Hz, 1H), 7.24-7.21 (m, 2H), 7.05-7.03 (m, 1H), 6.79 (d, J=8.0Hz, 1H), 5.64 (s, 1H), 4.97 (s, 2H), 1.37 (s, 6H).

2-Amino-N-(2-amino-5-(thiophen-2-yl)phenyl)-2-methylpropanamide (195)was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acid inScheme 5 with 2-((tert-butoxycarbonyl)amino)-2-methylpropanoic acid.ESI+MS: m/z 276 ([M+H]⁺); 1H NMR (500 MHz, d⁶-DMSO): δ 7.67 (s, 1H),7.35 (d, J=4.5 Hz, 1H), 7.22-7.20 (m, 2H), 7.04 (t, J=4.0 Hz, 1H), 6.79(d, J=8.5 Hz, 1H), 4.98 (s, 2H), 1.31 (s, 6H).

2-Amino-N-(2-amino-5-(thiophen-2-yl)phenyl)butanamide (196) was preparedby substituting tetrahydro-2H-pyran-4-carboxylic acid in Scheme 5 with2-((tert-butoxycarbonyl)amino)butanoic acid. ESI+MS: m/z 276 ([M+H]⁺);1H NMR (500 MHz, d⁶-DMSO): δ 9.59 (s, 1H), 7.34 (d, J=5.0 Hz, 1H),7.23-7.19 (m, 2H), 7.04 (t, J=4.0 Hz, 1H), 6.77 (d, J=8.5 Hz, 1H), 5.04(s, 2H), 3.30-3.27 (m, 2H), 1.74-0.167 (m, 1H), 1.55-1.49 (m, 1H), 0.94(t, J=7.0 Hz, 3H).

3-Acetamido-N-(2-amino-5-(thiophen-2-yl)phenyl)cyclobutanecarboxamide(14) was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acidin Scheme 5 with 3-acetamidocyclobutanecarboxylic acid. ESI+MS: m/z 330([M+H]+), 1H NMR (500 MHz, d6-DMSO): δ 9.10 (s, 1H), 8.16 (d, J=7.5 Hz,1H), 7.5 (s, 1H), 7.32 (d, J=5.0 Hz, 1H), 7.21-7.18 (m, 2H), 7.02 (t,J=4.0 Hz, 1H), 6.73 (d, J=8.5 Hz, 1H), 5.05 (s, 2H), 4.15 (q, J=9.0 Hz,1H), 2.91-2.88 (m, 1H), 2.38-2.35 (m, 2H), 2.10 (q, J=10.0 Hz, 2H), 1.75(s, 3H).

N-(2-aminophenyl)cyclohexanecarboxamide (21) was prepared bysubstituting tetrahydro-2H-pyran-4-carboxylic acid in Scheme 5 withcyclohexanecarboxylic acid and by substituting tert-butyl(2-amino-4-(thiophen-2-yl)phenyl)carbamate in Scheme 5 with tert-butyl(2-aminophenyl)carbamate. ESI+MS: m/z 319 ([M+H]+), 1H NMR (300 MHz,d6-DMSO): δ 9.02 (s, 1H), 7.15 (d, J=9 Hz, 1H), 6.89 (t, J=6 Hz, 1H),6.71 (d, J=6 Hz, 1H), 6.54 (t, J=6, 1H), 4.79 (bs, 2H), 1.87-1.72 (m,4H), 1.45-1.38 (m, 2H), 1.33-1.18 (m, 4H).

1-Acetyl-N-(2-aminophenyl)piperidine-4-carboxamide (36) was prepared bysubstituting tetrahydro-2H-pyran-4-carboxylic acid in Scheme 5 withpiperidine-1,4-dicarboxylic acid and by substituting tert-butyl(2-amino-4-(thiophen-2-yl)phenyl)carbamate in Scheme 5 with tert-butyl(2-aminophenyl)carbamate. ESI+MS: m/z 262 ([M+H]+), 1H NMR (500 MHz,d6-DMSO): δ 9.12 (s, 1H), 7.15 (d, J=6 Hz, 1H), 6.90 (t, J=9 Hz, 1H),6.72 (d, J=9 Hz, 1H), 6.54 (t, J=9 Hz, 1H), 4.82 (bs, 2H), 4.49-4.60 (m,2H), 3.90-3.75 (m, 2H), 3.30-3.00 (m, 2H), 2.40-2.30 (m, 1H), 2.01 (s,3H), 1.70-1.65 (m, 2H).

N-(2-amino-5-(5-methylthiophen-2-yl)phenyl)cyclohexanecarboxamide (19)was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acid inScheme 5 with cyclohexanecarboxylic acid and by substitutingthiophen-2-ylboronic acid in Scheme 2 with 5-methylthiophen-2-ylboronicacid. ESI+MS: m/z 315 ([M+H]+), 1H NMR (300 MHz, d6-DMSO): δ 9.07 (s,1H), 7.44 (d, J=3 Hz, 1H), 7.14 (dd, J=3 Hz, 6 Hz, 1H), 6.98 (d, J=3 Hz,1H), 6.75-6.64 (m, 2H0, 5.01 (bs, 1H0, 2.45-2.41 (m, 3H), 1.90-1.71 (m,4H), 1.70-1.63 (m, 1H), 1.50-1.38 (m, 2H0, 1.37-1.16 (m, 5H).

N-(2-amino-5-(5-methylthiophen-2-yl)phenyl)tetrahydro-2H-pyran-4-carboxamide(46) was prepared by substituting thiophen-2-ylboronic acid in Scheme 2with 5-methylthiophen-2-ylboronic acid. ESI+MS: m/z 315 ([M+H]+), 1H NMR(500 MHz, d6-DMSO): δ 9.19 (s, 1H0, 7.49 (d, J=3, 1H), 7.20 (dd, J=3 Hz,9 Hz, 1H), 3.60-6.70 (m, 2H), 5.08 (bs, 2H0, 4.01-3.90 (m, 2H),3.48-3.44 9m, 1H), 2.78-2.61 (m, 2H), 2.48 (s, 3H), 1.85-1.65 (m, 4H0,1.32-1.28 (m, 1H).

N-(2-amino-5-(4-methylthiophen-2-yl)phenyl)cyclohexanecarboxamide (20)was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acid inScheme 5 with cyclohexanecarboxylic acid and by substitutingthiophen-2-ylboronic acid in Scheme 2 with 4-methylthiophen-2-ylboronicacid. 1H NMR (500 MHz, d6-DMSO): δ 9.06 (s, 1H), 7.49 (d, J=3 Hz, 1 h),7.16 (dd, J=6 Hz, 1H), 7.03 (s, 1H), 6.92 (s, 1H0, 6.74 (d, J=6 Hz, 1H),5.06 (bs, 2H), 2.20 (s, 3H), 1.90-1.70 (m, 4H), 1.69-1.61 9m, 1H).

N-(2-amino-5-(4-methylthiophen-2-yl)phenyl)tetrahydro-2H-pyran-4-carboxamide(47) was prepared by substituting thiophen-2-ylboronic acid in Scheme 2with 4-methylthiophen-2-ylboronic acid. ESI+MS: m/z XXX ([M+H]+), 1H NMR(300 MHz, d6-DMSO): δ 9.12 (s, 1H), 7.49 (d, J=1 Hz, 1H), 7.18 (dd, J=1Hz, 9 Hz, 1H0, 7.04 (s, 1H), 6.92 (s, 1 h) m, 6.74 (d, J=9 Hz, 1H), 5.07(s, 2H), 4.00-3.85 (m, 2 h), 3.43-3.41 (m, 1H), 2.20 (s, 3 h), 1.80-1.60(m, 5H), 1.24 (bs, 1H).

N-(4-amino-[1,1′-biphenyl]-3-yl)tetrahydro-2H-pyran-4-carboxamide (49)was prepared by substituting thiophen-2-ylboronic acid in Scheme 2 withphenyl boronic acid. ESI+MS: m/z 297 ([M+H]+), 1H NMR (500 MHz,d6-DMSO): δ 91.5 (s, 1H), 7.60-7.45 (m, 3H), 7.44-7.36 (m, 2H),7.30-7.20 (m, 2H), 6.81 (d, J=6 Hz, 1H), 5.02 (s, 1H), 3.93 (d, J=9 Hz,2H), 3.43-3.36 (m, 2H), 1.80-1.60 (m, 4H).

N-(2-amino-5-(thiophen-2-yl)phenyl)-3-methyl-3-azabicyclo[3.1.0]hexane-6-carboxamide(58) was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acidin Scheme 5 with 3-methyl-3-azabicyclo[3.1.0]hexane-6-carboxylic acid.ESI+MS: m/z 314 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 9.31 (s, 1H),7.60 (d, J=1.0 Hz, 1H), 7.33 (d, J=5.0 Hz, 1H), 7.22-7.15 (m, 2H), 7.03(t, J=4.5 Hz, 1H), 6.73 (d, J=8.5 Hz, 1H), 5.09 (s, 2H), 3.04-2.95 (m,2H), 2.38-2.20 (m, 6H), 1.90-1.80 (m, 2H).

3-acetyl-N-(2-amino-5-(thiophen-2-v)phenyl)-3-azabicyclo[3.1.0]hexane-6-carboxamide(57) was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acidin Scheme 5 with 3-acetyl-3-azabicyclo[3.1.0]hexane-6-carboxylic acid.ESI+MS: m/z 342 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 9.35 (s, 1H),7.59 (d, J=2.0 Hz, 1H), 7.34 (d, J=5.0 Hz, 1H), 7.24-7.16 (m, 2H), 7.03(t, J=4.0 Hz, 1H), 6.74 (d, J=8.5 Hz, 1H), 5.11 (s, 2H), 3.75-3.60 (m,3H), 3.35-3.30 (m, 1H), 2.12-1.98 (m, 2H), 1.93 (s, 3H), 1.71-1.67 (m,1H).

3-(1-Acetylpiperidin-4-yl)-N-(2-amino-5-(thiophen-2-yl)phenyl)propanamide(164) was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acidin Scheme 5 with 3-(1-acetylpiperidin-4-yl)propanoic acid. ESI+MS: m/z372 ([M]⁺); 1HNMR (500 MHz, d⁶-DMSO): δ 9.15 (s, 1H), 7.49 (s, 1H), 7.34(d, J=5.5 Hz, 1H), 7.22-7.19 (m, 2H), 7.03 (t, J=4.5 Hz, 1H), 6.74 (d,J=8.5 Hz, 1H), 5.06 (s, 2H), 4.36 (d, J=13.0 Hz, 1H), 3.79 (d, J=13.0Hz, 1H), 2.98 (t, J=12.5 Hz, 1H), 2.37 (t, J=7.5 Hz, 2H), 1.97 (s, 3H),1.71 (t, J=14.0 Hz, 2H), 1.57-1.50 (m, 3H), 1.09-0.93 (m, 2H).

3-Acetamido-N-(2-amino-5-(thiophen-2-yl)phenyl)cyclobutanecarboxamide(14) was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acidin Scheme 5 with 3-acetamidocyclobutanecarboxylic acid. ESI+MS: m/z 330([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 9.10 (s, 1H), 8.16 (d, J=7.5 Hz,1H), 7.5 (s, 1H), 7.32 (d, J=5.0 Hz, 1H), 7.21-7.18 (m, 2H), 7.02 (t,J=4.0 Hz, 1H), 6.73 (d, J=8.5 Hz, 1H), 5.05 (s, 2H), 4.15 (q, J=9.0 Hz,1H), 2.91-2.88 (m, 1H), 2.38-2.35 (m, 2H), 2.10 (q, J=10.0 Hz, 2H), 1.75(s, 3H).

N-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)-3-oxabicyclo[3.1.0]hexane-6-carboxamide(204) was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acidin Scheme 5 with tert-butyl3-acetyl-3-azabicyclo[3.1.0]hexane-6-carboxylate and tert-butyl(2-amino-4-(thiophen-2-yl)phenyl)carbamate in Scheme 5 with tert-butyl(3-amino-4′-fluoro-[1,1′-biphenyl]-4-yl)carbamate. ESI+MS: m/z 313([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 9.38 (s, 1H), 7.58 (s, 1H),7.53-7.50 (m, 2H), 7.21-7.18 (m, 3H), 6.79 (d, J=8.5 Hz, 1H), 5.04 (s,2H), 3.85 (d, J=8.5 Hz, 2H), 3.67 (d, J=8.5 Hz, 2H), 2.08 (s, 2H), 1.77(m, 1H).

N-(2-aminophenyl)cyclohex-1-enecarboxamide (244) was prepared bysubstituting tetrahydro-2H-pyran-4-carboxylic acid in Scheme 5 withcyclohex-1-enecarboxylic acid and tert-butyl(2-amino-4-(thiophen-2-yl)phenyl)carbamate in Scheme 5 with tert-butyl(2-aminophenyl)carbamate. ESI+MS: m/z 217 ([M+H]⁺), 1H NMR (400 MHz,d⁶-DMSO): δ 8.98 (s, 1H), 7.14-7.12 (m, 1H), 6.98-6.94 (m, 1H), 6.79(dd, J_(1,2)=1.2 Hz, J_(1,3)=8.0 Hz, 2H), 6.62-6.58 (m, 1H), 4.80 (s,2H), 2.31 (t, J=2.0 Hz, 2H), 2.22-2.21 (m, 2H), 1.69-1.60 (m, 4H).

N-(2-amino-5-(thiophen-2-yl)phenyl)-8-oxabicyclo[3.2.1]octane-3-carboxamide(182) was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acidin Scheme 5 with 8-oxabicyclo[3.2.1]octane-3-carboxylic acid. ESI+MS:m/z 329 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 9.07 (s, 1H), 7.48 (s,1H), 7.34 (d, J=5.0 Hz, 1H), 7.22-7.19 (m, 2H), 7.03 (t, J=4.0 Hz, 1H),6.74 (d, J=8.5 Hz, 1H), 5.04 (s, 2H), 4.34 (bs, 2H), 2.91-2.89 (m, 1H),1.86-1.76 (m, 6H), 1.62-1.58 (m, 2H).

N-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)-8-oxabicyclo[3.2.1]octane-3-carboxamide(191) was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acidin Scheme 5 with 8-oxabicyclo[3.2.1]octane-3-carboxylic acid andtert-butyl (2-amino-4-(thiophen-2-yl)phenyl)carbamate in Scheme 5 withtert-butyl (3-amino-4′-fluoro-[1,1′-biphenyl]-4-yl)carbamate. ESI+MS:m/z 341 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 9.05 (s, 1H), 7.53-7.49(m, 3H), 7.21-7.18 (m, 3H), 6.79 (d, J=8.5 Hz, 1H), 4.98 (s, 2H), 4.34(s, 2H), 2.90-2.89 (m, 1H), 1.87-1.77 (m, 6H), 1.60 (dd, J=4.5 Hz,J=12.5 Hz, 2H).

3-(1-acetylpiperidin-4-yl)-N-(2-aminophenyl)propanamide (206) wasprepared by substituting tetrahydro-2H-pyran-4-carboxylic acid in Scheme5 with 3-(1-(tert-butoxycarbonyl)piperidin-4-yl)propanoic acid andtert-butyl (2-amino-4-(thiophen-2-yl)phenyl)carbamate in Scheme 5 withtert-butyl (2-aminophenyl)carbamate. ESI+MS: m/z 290 ([M+H]⁺), 1H NMR(300 MHz, d⁶-DMSO): δ 9.12 (s, 1H), 7.15 (d, J=9 Hz, 1H), 6.89 (t, J=9Hz, 1H), 6.72 (t, J=9 Hz, 1H), 4.35 (d, J=45 Hz, 1H), 3.79 (d, J=45 Hz,1H), 3.01-2.93 (m, 2H), 2.36-2.32 (m, 2H), 1.97 (s, 3H), 1.74-1.66 (m,2H), 1.57-1.50 (m, 3H), 1.08-0.91 (m, 2H).

N-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)-6-oxospiro[3.3]heptane-2-carboxamide(208) was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acidin Scheme 5 with 1,6-oxospiro[3.3]heptane-2-carboxylic acid andtert-butyl (2-amino-4-(thiophen-2-yl)phenyl)carbamate in Scheme 5 withtert-butyl (3-amino-4′-fluoro-[1,1′-biphenyl]-4-yl)carbamate. ESI+MS:m/z 340 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 7.80 (bs, 1H), 7.55-7.51(m, 2H), 7.37 (d, J=2.5 Hz, 1H), 7.20 (t, J=11.0 Hz, 3H), 6.79 (d,J=10.5 Hz, 1H), 5.15 (bs, 2H), 4.14 (s, 4H), 3.33 (s, 4H).

N-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)-2,3-dihydro-1H-indene-2-carboxamide(214) was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acidin Scheme 5 with 2,3-dihydro-1H-indene-2-carboxylic acid and tert-butyl(2-amino-4-(thiophen-2-yl)phenyl)carbamate in Scheme 5 with tert-butyl(3-amino-4′-fluoro-[1,1′-biphenyl]-4-yl)carbamate. ESI+MS: m/z 346([M+H]⁺), 1H NMR (400 MHz, d⁶-DMSO): δ 9.30 (s, 1H), 7.54 (dd,J_(1,2)=5.6 Hz, J_(1,3)=8.4 Hz, 3H), 7.23-7.13 (m, 7H), 6.81 (d, J=8.4Hz, 1H), 5.04 (s, 2H), 3.52-3.43 (m, 1H), 3.20 (d, J=8.8 Hz, 4H).

N-(2-amino-4-fluorophenyl)cyclopent-1-enecarboxamide (207) was preparedby substituting tetrahydro-2H-pyran-4-carboxylic acid in Scheme 5 withcyclopent-1-enecarboxylic acid and tert-butyl(2-amino-4-(thiophen-2-yl)phenyl)carbamate in Scheme 5 with tert-butyl2-amino-5-fluorophenylcarbamate. ESI+MS: m/z 321 ([M+H]⁺), 1H NMR (300MHz, d⁶-DMSO): δ 8.98 (s, 1H), 7.02 (dd, J₁=6 Hz, J₂=3 Hz, 1H), 6.66(bs, 1H), 6.50 (dd, J₁=3 Hz, J₂=12 Hz, 1H), 6.32 (dt, J=3 Hz, J₂=9 Hz,1H), 5.13 (s, 2H), 2.59-2.54 (m, 3H), 1.95-1.84 (m, 3H).

N-(2-amino-5-(pyridin-4-yl)phenyl)-1-methyl-2-oxopiperidine-4-carboxamide(205) was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acidin Scheme 5 with 1-methyl-2-oxopiperidine-4-carboxylic acid andtert-butyl (2-amino-4-(thiophen-2-yl)phenyl)carbamate in Scheme 5 withtert-butyl (2-amino-4-(pyridin-4-yl)phenyl)carbamate. ESI+MS: m/z 325([M+H]+); 1H NMR (500 MHz, d6-DMSO): δ 9.24 (s, 1H), 8.50 (d, J=5.0 Hz,2H), 7.69 (s, 1H), 7.53 (d, J=5.0 Hz, 2H), 7.43 (d, J=7.5 Hz, 1H), 6.83(d, J=8.5 Hz, 1H), 5.30 (s, 2H), 3.35 (m, 2H), 2.95 (s, 1H), 2.82 (s,3H), 2.44-2.41 (m, 2H), 2.09 (m, 1H), 1.89 (m, 1H).

N-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)chroman-3-carboxamide (211)was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acid inScheme 5 with chroman-3-carboxylic acid and tert-butyl(2-amino-4-(thiophen-2-yl)phenyl)carbamate in Scheme 5 with tert-butyl(3-amino-4′-fluoro-[1,1′-biphenyl]-4-yl)carbamate. ESI+MS: m/z 363([M+H]+); ¹H NMR (400 MHz, DMSO-d6): δ 9.41 (s, 1H), 7.53 (dd, J=5.6 Hz,J=8.8 Hz, 3H), 7.25-7.07 (m, 5H), 6.87-6.78 (m, 3H), 5.07 (s, 2H), 4.50(d, J=10.8 Hz, 1H), 4.00 (t, J=10.0 Hz, 1H), 3.08-2.99 (m, 3H).

N-(2-aminophenyl)-4-fluoro-2,3-dihydro-1H-indene-2-carboxamide (219) wasprepared by substituting tetrahydro-2H-pyran-4-carboxylic acid in Scheme5 with 4-fluoro-2,3-dihydro-1H-indene-2-carboxylic acid and tert-butyl(2-amino-4-(thiophen-2-yl)phenyl)carbamate in Scheme 2 with tert-butyl(2-aminophenyl)carbamate. ESI+MS: m/z 271 ([M+H]+); ¹H NMR (400 MHz,D6-DMSO-d₆): δ 9.26 (s, 1H), 7.22-7.17 (m, 2H), 7.07 (d, J=7.6 Hz, 1H),6.98-6.88 (m, 2H), 6.73 (dd, J=1.2 Hz, J=8.0 Hz, 1H), 6.56-6.52 (m, 1H),4.84 (s, 2H), 3.56-3.47 (m, 1H), 3.31-3.11 (m, 4H).

N-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)-2,3-dihydro-1H-indene-1-carboxamide(221) was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acidin Scheme 5 with 2,3-dihydro-1H-indene-1-carboxylic acid and tert-butyl(2-amino-4-(thiophen-2-yl)phenyl)carbamate in Scheme 5 with tert-butyl(3-amino-4′-fluoro-[1,1′-biphenyl]-4-yl)carbamate. ESI+MS: m/z 347([M+H]+); 1H NMR (400 MHz, d6-DMSO): δ 9.61 (s, 1H), 7.55-7.52 (m, 3H),7.41-7.39 (m, 1H), 7.27-7.18 (m, 6H), 6.86 (d, J=8.0 Hz, 1H), 4.18 (t,J=7.6 Hz, 1H), 3.06-3.03 (m, 1H), 2.92-2.90 (m, 1H), 2.36-2.31 (m, 2H).

N-(4-amino-3′-fluoro-[1,1′-biphenyl]-3-yl)tetrahydro-2H-pyran-4-carboxamide(225) was prepared by substituting tert-butyl(2-amino-4-(thiophen-2-yl)phenyl)carbamate in Scheme 5 with tert-butyl(3-amino-3′-fluoro-[1,1′-biphenyl]-4-yl)carbamate. ESI+MS: m/z 315([M+H]+); 1HNMR (400 MHz, d6-DMSO): δ 9.11 (s, 1H), 7.56 (br s, 3H),7.40-7.26 (m, 4H), 7.02 (t, J=7.6 Hz, 1H), 6.78 (d, J=8.4 Hz, 1H), 5.07(s, 2H), 3.90 (d, J=11.2 Hz, 2H), 3.37-3.35 (m, 2H), 2.65-2.64 (m, 1H),1.75-1.65 (m, 4H).

N-(2-amino-5-(pyridin-4-yl)phenyl)-3-fluorocyclobutanecarboxamide (239)was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acid inScheme 5 with 3-fluorocyclobutanecarboxylic acid and tert-butyl(2-amino-4-(thiophen-2-yl)phenyl)carbamate in Scheme 5 with tert-butyl(2-amino-4-(pyridin-4-yl)phenyl)carbamate. ESI+MS: m/z 286 (M+H); 1H NMR(300 MHz, d6-DMSO): δ 9.24 (s, 1H), δ 8.50 (d, J=6 Hz, 2H), δ 7.72 (bs,1H), δ 7.54 (d, J=6 Hz, 1H), δ 7.43 (d, J=6 Hz, 1H), δ 6.83 (d, J=9 Hz,1H), δ 5.31 (s, 2H), δ 5.16 (tm, J=69 Hz, 1H), δ 3.35-3.22 (m, 1H), δ2.62-2.50 (m, 4H).

(E)-3-(1-acetylpiperidin-4-yl)-N-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)acrylamide(242) was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acidin Scheme 5 with (E)-3-(1-acetylpiperidin-4-yl)acrylic acid andtert-butyl (2-amino-4-(thiophen-2-yl)phenyl)carbamate in Scheme 5 withtert-butyl (3-amino-4′-fluoro-[1,1′-biphenyl]-4-yl)carbamate. ESI+MS:m/z 382 ([M+H]+); 1H NMR (400 MHz, d6-DMSO): δ 9.29 (s, 1H), 7.60-7.51(m, 3H), 7.23-7.18 (m, 3H), 6.18 (d, J=15.6 Hz, 1H), 5.06 (s, 2H), 4.39(d, J=13.2 Hz, 1H), 3.84 (d, J=14.0 Hz, 1H), 3.12-3.06 (m, 1H),2.66-2.49 (m, 2H), 2.00 (s, 3H), 1.76 (t, J=13.6 Hz, 2H), 1.35-1.29 (m,1H), 1.23-1.15 (m, 1H).

N-(2-aminophenyl)-2,3-dihydro-1H-indene-2-carboxamide (223) was preparedby substituting tetrahydro-2H-pyran-4-carboxylic acid in Scheme 5 with2,3-dihydro-1H-indene-2-carboxylic acid and by substituting tert-butyl(2-amino-4-(thiophen-2-yl)phenyl)carbamate in Scheme 5 with tert-butyl(2-aminophenyl)carbamate. ESI+MS: m/z 253 ([M+H]⁺), 1H NMR (300 MHz,d6-DMSO): δ 9.26 (s, 1H), 7.24-7.12 (m, 5H), 6.90 (t, J=6 Hz, 1H), 6.73(d, J=6 Hz, 1H), 6.55 (t, J=6 Hz, 1H), 4.85 (s, 2H), 3.45 (quint, J=9Hz, 1H), 3.16 (d, J=9 Hz, 4H).

3-(1-acetylpiperidin-4-yl)-N-(2-amino-4-fluorophenyl)propanamide (243)was prepared by substituting tetrahydro-2H-pyran-4-carboxylic acid inScheme 5 with 3-(1-acetylpiperidin-4-yl)propanoic acid and bysubstituting tert-butyl (2-amino-4-(thiophen-2-yl)phenyl)carbamate inScheme 5 with tert-butyl 2-amino-5-fluorophenylcarbamate. ESI+MS: m/z408 ([M+H]⁺).

N-(2-amino-4-fluorophenyl)-2,3-dihydro-1H-indene-2-carboxamide (237) wasprepared by substituting tetrahydro-2H-pyran-4-carboxylic acid in Scheme5 with 2,3-dihydro-1H-indene-2-carboxylic acid and by substitutingtert-butyl (2-amino-4-(thiophen-2-yl)phenyl)carbamate in Scheme 5 withtert-butyl 2-amino-5-fluorophenylcarbamate. ESI+MS: m/z 271 ([M+H]⁺), 1HNMR (400 MHz, d6-DMSO): δ 9.16 (bs, 1H), 7.21-7.13 (m, 5H), 6.51-6.31(m, 2H), 5.15 (s, 2H), 3.42-3.40 (m, 1H), 3.31-3.17 (m, 4H).

Synthesis of N-(2-amino-5-(thiophen-2-yl)phenyl)isobutyramide (4)

To a solution of tert-butyl (2-amino-4-(thiophen-2-yl)phenyl)carbamate(200 mg, 0.689 mmol) in dichloromethane (4 mL) was added isobutyrylchloride (0.08 mL, 0.76 mmol, 1 equiv.) and TEA (0.23 mL, 1.72 mmol, 2.5equiv.) at 0° C. The reaction was warmed to room temperature and stirredfor 16 h. The reaction was then diluted with dichloromethane and water.The organic layers were washed with water and brine, dried over Na₂SO₄and concentrated. The crude material was purified by columnchromatography (silica gel, EtOAc/hexanes) to afford tert-butyl(2-isobutyramido-4-(thiophen-2-yl)phenyl)carbamate (0.2 g, 81% yield).

To a stirred solution of tert-butyl(2-isobutyramido-4-(thiophen-2-yl)phenyl)carbamate (0.2 g, 0.52 mmol) inmethanol (4 mL) was added 4M HCl in Dioxane (2 mL, 0.53 mmol, 1 equiv.)at 0° C. The reaction was warmed to room temperature and stirred for 2h. After completion the reaction was concentrated under reduced pressurethen basified with a saturated aqueous solution of NaHCO₃. The obtainedsolid was filtered and dried. The crude material was purified by columnchromatography (silica gel, MeOH/CH₂Cl₂) to affordN-(2-amino-5-(thiophen-2-yl)phenyl)isobutyramide (0.05 mg, 36% yield).ESI+MS: m/z 261 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 9.10 (s, 1H0, 7.5(s, 1H0, 7.34 (d, J=4.5 Hz, 1H), 7.23-7.19 (m, 2H), 7.05-7.03 (m, 1H0,6.75 (d, J=8.5 hz, 1H0, 5.03 (s, 2H), 2.67-2.64 (m, 1H), 1.13 (d, J=7Hz, 6H).

One skilled in the art will recognize that other compounds describedbelow were prepared in a similar manner to the procedures describedabove.

N-(2-amino-5-(thiophen-2-yl)phenyl)acetamide (3) was prepared bysubstituting isobutyryl chloride in Scheme 6A with acetyl chloride.ESI+MS: m/z 233 ([M+H]⁺), 1H NMR (300 MHz, d⁶-DMSO): δ 9.23 (s, 1H),7.51 (d, J=3.0 Hz, 1H), 7.35 (d, J=6.0 Hz, 1H), 7.26-7.16 (m, 2H), 7.05(dd, J=3.0, 4.0 Hz, 1H), 6.76 (d, J=9.0 Hz, 1H), 5.14 (bs, 2H), 2.08 (s,3H).

N-(4-amino-[1,1′-biphenyl]-3-yl)acetamide (2) was prepared bysubstituting isobutyryl chloride in Scheme 6A with acetyl chloride andby substituting tert-butyl (2-amino-4-(thiophen-2-yl)phenyl)carbamatewith tert-butyl (3-amino-[1,1′-biphenyl]-4-yl)carbamate. ESI+MS: m/z 227([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 9.21 (s, 1H), 7.55-7.50 (m, 3H),7.39 (t, J=7.5 Hz, 2H), 7.27-7.21 (m, 2H), 6.81 (d, J=8.5 Hz, 1H), 5.12(s, 2H), 2.07 (s, 3H).

N-(2-amino-5-(thiophen-2-yl)phenyl)pivalamide (5) was prepared bysubstituting isobutyrylchloride in Scheme 6A with pivaloyl chloride.ESI+MS: m/z 275 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 8.84 9s, 1H),7.35-7.32 (m, 1H), 7.30-7.22 9m, 2H), 7.21-7.19 (m, 1H), 7.06-7.01 (m,1H), 6.78 (d, J=8 Hz, 1H), 4.89 (s, 2H), 1.25 (s, 9H).

N-(2-amino-5-(thiophen-2-yl)phenyl)-3,3-dimethylbutanamide (6) wasprepared by substituting isobutyrylchloride in Scheme 6A with3,3-dimethylbutanoyl chloride. ESI+MS: m/z 289 ([M+H]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 9.15 (s, 1H), 7.47-7.46 (m, 1H), 7.36-7.34 (m, 1H),7.24-7.19 (m, 2H), 7.06-7.03 (m, 1H0, 6.76 (d, J=8 Hz, 1H), 5.04 (s,2H), 2.22 (s, 2H), 1.05 (s, 9H).

N-(2-amino-5-(pyrazin-2-yl)phenyl)cyclohexanecarboxamide (27) wasprepared by substituting isobutyryl chloride in Scheme 6A withcyclohexanecarbonyl chloride and by substituting tert-butyl(2-amino-4-(thiophen-2-yl)phenyl)carbamate with tert-butyl(2-amino-4-(pyrazin-2-yl)phenyl)carbamate. ESI+MS: m/z 297 ([M+H]⁺), 1HNMR (500 MHz, d⁶-DMSO): δ 9.09 (s, 1H), 9.03 (d, J=1.5 Hz, 1H),8.58-8.55 (m, 1H), 8.41 (d, J=3.0 Hz, 1H), 8.04 (d, J=2.0 Hz, 1H), 7.72(dd, J=12.5, 2.0 Hz, 1H), 6.82 (d, J=12.5 Hz, 1H), 5.33 (bs, 2H),2.46-2.35 (m, 1H), 1.90-1.80 (m, 2H), 1.80-1.72 (m, 2H), 1.72-1.60 (m,2H), 1.50-1.38 (m, 2H), 1.38-1.16 (m, 3H).

N-(2-amino-5-(pyridin-3-yl)phenyl)cyclohexanecarboxamide (22) wasprepared by substituting isobutyryl chloride in Scheme 6A withcyclohexanecarbonyl chloride and by substituting tert-butyl(2-amino-4-(thiophen-2-yl)phenyl)carbamate with tert-butyl(2-amino-4-(pyridin-3-yl)phenyl)carbamate. ESI+MS: m/z 296 ([M+H]⁺), 1HNMR (500 MHz, d⁶-DMSO): δ 9.10 (s, 1H), 8.75 (s, 1H), 8.44 (d, J=3.5 Hz,1H), 7.89 (d, J=7.5 Hz, 1H), 7.59 (s, 1H), 7.42-7.36 (m, 1H), 7.29 (d,J=8.0 Hz, 1H), 6.83 (d, J=8.0 Hz, 1H), 5.10 (bs, 2H), 2.44-2.36 (m, 1H),1.88-1.82 (m, 2H), 1.79-1.74 (m, 2H), 1.68-1.64 (m, 1H), 1.50-1.39 (m,2H), 1.31-1.20 (m, 3H).

N-(4-amino-2′,3′,4′,5′-tetrahydro-[1,1′-biphenyl]-3-yl)cyclohexanecarboxamide(26) was prepared by substituting isobutyryl chloride in Scheme 6A withcyclohexanecarbonyl chloride and by substituting tert-butyl(2-amino-4-(thiophen-2-yl)phenyl)carbamate in Scheme 6A with tert-butyl(3-amino-2′,3′,4′,5′-tetrahydro-[1,1′-biphenyl]-4-yl)carbamate. ESI+MS:m/z 299 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 8.98 (s, 1H), 7.29 (s,1H), 6.96 (d, J=8.5 Hz, 1H), 6.65 (d, J=8.5 Hz, 1H), 5.90 (bs, 1H), 4.77(s, 2H), 2.40-2.32 (m, 1H), 2.30-2.22 (m, 2H), 2.16-2.08 (m, 2H),1.86-1.73 (m, 4H), 1.72-1.61 (m, 3H), 1.60-1.54 (m, 2H), 1.46-1.36 (m,2H), 1.34-1.14 (m, 3H).

N-(2-amino-5-vinylphenyl)cyclohexanecarboxamide (23) was prepared bysubstituting isobutyryl chloride in Scheme 6A with cyclohexanecarbonylchloride and by substituting tert-butyl(2-amino-4-(thiophen-2-yl)phenyl)carbamate in Scheme 6A with tert-butyl(2-amino-4-vinylphenyl)carbamate. ESI+MS: m/z 245 ([M+H]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 7.28 (d, J=1.5 Hz, 1H), 7.03 (dd, J=2 Hz, 8.5 Hz, 1H),6.67 (d, J=8.5 Hz, 1H), 6.56-6.49 (m, 1H), 5.46 (d, J=17 Hz, 1H),4.98-4.94 (m, 3H), 2.40-2.33 (m, 1H), 1.80 (d, J=13 Hz, 2H), 1.77-1.73(m, 2H), 1.65 (d, J=12 Hz, 1H), 1.45-1.36 (m, 2H), 1.31-1.18 (m, 3H).

N-(4-amino-2′-methyl-[1,1′-biphenyl]-3-yl)cyclohexanecarboxamide (24)was prepared by substituting isobutyryl chloride in Scheme 6A withcyclohexanecarbonyl chloride and by substituting tert-butyl(2-amino-4-(thiophen-2-yl)phenyl)carbamate in Scheme 6A with tert-butyl(3-amino-2′-methyl-[1,1′-biphenyl]-4-yl)carbamate. ESI+MS: m/z 309([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 9.06 (s, 1H), 7.24-7.18 (m, 1H),7.14-7.12 (m, 1H), 6.90-6.88 (m, 1H), 6.78 (d, J=8 Hz, 1H), 4.92 (s,2H), 2.52-2.50 (m, 1H), 2.25 (s, 3H), 1.83 (d, J=12.5 Hz, 2H), 1.76 (d,J=11 Hz, 2H), 1.67-1.64 (m, 1H), 1.46-1.37 (m, 2H), 1.31-1.18 (m, 3H).

N-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)cyclohexanecarboxamide (25)was prepared by substituting isobutyryl chloride in Scheme 6A withcyclohexanecarbonyl chloride and by substituting tert-butyl(2-amino-4-(thiophen-2-yl)phenyl)carbamate in Scheme 6A with tert-butyl(3-amino-4′-fluoro-[1,1′-biphenyl]-4-yl)carbamate. ESI+MS: m/z 313([M+H]⁺), 1H NMR (500 MHz, d6-DMSO): δ 9.05 (s, 1H), 7.53-7.51 (m, 3H),7.21-7.20 (m, 3H), 6.79 (d, J=8.5 Hz, 1H), 4.97 (s, 2H), 2.39 (t, J=11.0Hz, 1H), 1.85-1.75 (m, 4H), 1.65 (d, J=11.0 Hz, 2H), 1.46-1.39 (m, 2H),1.33-1.17 (m, 2H).

Example 2

Synthesis of propyl azetidin-3-ylmethyl(2-amino-5-(thiophen-2-yl)phenyl)carbamate (75)

To a stirred solution of 5-bromo-2-nitroaniline (10.3 g, 47.3 mmol, 1eq.), thiophen-2-ylboronic acid (9.08 g, 70.9 mmol, 1.5 eq.) andtetrakis(triphenylphosphine)palladium(0) (5.46 g, 4.73 mmol, 0.1 eq.) inTHF (150 mL) was added a solution of sodium carbonate (7.27 g, 68.6mmol, 1.45 eq.) in water (15 mL). The resulting mixture was warmed to90° C. for 18 h. The reaction was then diluted with EtOAc and water andfiltered through Celite. The organic layer was separated and dried oversodium sulfate, filtered and concentrated under reduced pressure. Thecrude product was purified by column chromatography (silica gel, 2%EtOAc/hexanes) to afford 2-nitro-5-(thiophen-2-yl)aniline (12.0 g, 79%yield).

To a solution of 2-nitro-5-(thiophen-2-yl)aniline (0.30 g, 1.36 mmol, 1eq.) and triphosgene (0.24 g, 0.82 mmol, 0.6 eq.) in dichloromethane (7mL) at 0° C. was added TEA (0.38 mL, 2.90 mmol, 2 eq.). The reaction wasstirred at room temperature for 2 h, then cooled down to 0° C.Tert-butyl 3-(hydroxymethyl)azetidine-1-carboxylate (0.31 g, 1.64 mmol,1.2 eq.) in dichloromethane (7 mL) and TEA (0.38 mL, 2.90 mmol, 2 eq.)were added at 0° C. The reaction was warmed to room temperature andstirred for 1 h. The reaction was diluted with dichloromethane andwashed with citric acid solution then brine. The organic layer wasseparated, dried over sodium sulfate and concentrated. The crude productwas purified by column chromatography (silica gel, 25% EtOAc/hexanes) toobtain tert-butyl3-((((2-nitro-5-(thiophen-2-yl)phenyl)carbamoyl)oxy)methyl)azetidine-1-carboxylate(0.59 g, 85% yield).

To a stirred solution of tert-butyl3-((((2-nitro-5-(thiophen-2-yl)phenyl)carbamoyl)oxy)methyl)azetidine-1-carboxylate(0.14 g, 0.32 mmol, 1.0 eq.) in MeOH (10 mL) was added Pd/C (40 mg, 0.38mmol, 1.2 eq.). The reaction mixture was stirred at room temperatureunder H₂ atmosphere for 1 h. The reaction mixture was then filteredthrough Celite, the solids washed with methanol. The filtrate wasconcentrated under reduced pressure to afford pure amine (0.12 g, 89%yield) which was used in the next step without further purification.

To a solution of tert-butyl3-((((2-amino-5-(thiophen-2-yl)phenyl)carbamoyl)oxy)methyl)azetidine-1-carboxylate(0.12 g, 0.30 mmol) in dichloromethane (5 mL) was added TFA (1.5 mL) at0° C. The reaction was then warmed to room temperature and stirred for 2h. The solvents were then removed under reduced pressure. A saturatedsolution of sodium bicarbonate was added. The product was extracted withethyl acetate, washed with water and brine. The combined organic layerswere dried over sodium sulfate, filtered and concentrated under reducedpressure. The crude solid was washed with ether to obtainazetidin-3-ylmethyl (2-amino-5-(thiophen-2-yl)phenyl)carbamate (49.6 mg,55% yield). ESI+MS: m/z 304 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 8.78(bs, 1H), 7.53 (bs, 1H), 7.34 (d, J=4.5 Hz, 1H), 7.22-7.16 (m, 2H), 7.04(dd, J=5.5; 4.0 Hz, 1H), 6.76-6.70 (m, 1H), 5.18 (bs, 2H), 4.23 (d,J=6.0 Hz, 2H), 3.92 (t, J=8.5 Hz, 2H), 3.70-3.64 (m, 2H), 3.08-3.00 (m,1H).

One skilled in the art will recognize that other compounds describedbelow can be prepared in a similar manner to the procedures describedabove.

Propyl (2-amino-5-(thiophen-2-yl)phenyl)carbamate (76) was prepared bysubstituting tert-butyl 3-(hydroxymethyl)azetidine-1-carboxylate inScheme 8 with propanol. ESI+MS: m/z 277 ([M+H]⁺), 1H NMR (500 MHz,d⁶-DMSO): δ 8.60 (bs, 1H), 7.52 (bs, 1H), 7.34 (d, J=5.0 Hz, 1H),7.21-7.16 (m, 2H), 7.05-7.02 (m, 1H), 6.71 (d, J=8.5 Hz, 1H), 5.11 (s,2H), 4.02 (t, J=7.0 Hz, 2H), 1.68-1.60 (m, 2H), 0.94 (t, J=7.5 Hz, 3H).

Pyridin-3-ylmethyl (2-amino-5-(thiophen-2-yl)phenyl)carbamate (86) wasprepared by substituting tert-butyl3-(hydroxymethyl)azetidine-1-carboxylate in Scheme 8 withpyridin-3-ylmethanol. ESI+MS: m/z 326 ([M+H]⁺), 1H NMR (500 MHz,d⁶-DMSO): δ 8.80 (bs, 1H), 8.66 (s, 1H), 8.55 (d, J=3.5 Hz, 1H),7.80-7.73 (m, 1H), 7.53 (bs, 1H), 7.45-7.40 (m, 1H), 7.34 (d, J=5.0 Hz,1H), 7.22-7.16 (m, 2H), 7.05-7.00 (m, 1H), 6.72 (d, J=8.0 Hz, 1H), 5.19(s, 2H), 5.16 (s, 2H).

Pyridin-2-ylmethyl (2-amino-5-(thiophen-2-yl)phenyl)carbamate (87) wasprepared by substituting tert-butyl3-(hydroxymethyl)azetidine-1-carboxylate in Scheme 8 withpyridin-2-ylmethanol. ESI+MS: m/z 326 ([M+H]⁺), 1H NMR (500 MHz,d⁶-DMSO): δ 8.89 (bs, 1H), 8.57 (d, J=4.0 Hz, 1H), 7.84 (t, J=7.5 Hz,1H), 7.55 (bs, 1H), 7.52-7.44 (m, 1H), 7.38-7.32 (m, 2H), 7.24-7.16 (m,2H), 7.03 (dd, J=3.5, 5.0 Hz, 1H), 6.73 (d, J=8.5 Hz, 1H), 5.21 (s, 2H),5.19 (s, 2H).

Pyridin-4-ylmethyl (2-amino-5-(thiophen-2-yl)phenyl)carbamate (88) wasprepared by substituting tert-butyl3-(hydroxymethyl)azetidine-1-carboxylate in Scheme 8 withpyridin-4-ylmethanol. ESI+MS: m/z 326 ([M+H]⁺), 1H NMR (500 MHz,d⁶-DMSO): δ 8.91 (bs, 1H), 8.58 (d, J=4.0 Hz, 2H), 7.53 (bs, 1H),7.46-7.38 (m, 2H), 7.34 (d, J=5.0 Hz, 1H), 7.24-7.16 (m, 2H), 7.03 (dd,J=4.0, 5.0 Hz, 1H), 6.74 (d, J=8.5 Hz, 1H), 5.20 (s, 2H), 5.18 (s, 2H).

(Tetrahydro-2H-pyran-4-yl)methyl(2-amino-5-(thiophen-2-yl)phenyl)carbamate (91) was prepared bysubstituting tert-butyl 3-(hydroxymethyl)azetidine-1-carboxylate inScheme 8 with (tetrahydro-2H-pyran-4-yl)methanol. ESI+MS: m/z 333([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 8.61 (bs, 1H), 7.52 (bs, 1H),7.34 (d, J=5.0 Hz, 1H), 7.20-7.16 (m, 2H), 7.03 (dd, J=3.5, 5.0 Hz, 1H),6.72 (d, J=8.0 Hz, 1H), 5.12 (s, 2H), 3.94 (d, J=7.0 Hz, 2H), 3.90-3.82(m, 2H), 3.35-3.28 (m, 2H), 1.95-1.85 (m, 1H), 1.64-1.56 (m, 2H),1.34-1.20 (m, 2H).

Cyclopentylmethyl (2-amino-5-(thiophen-2-yl)phenyl)carbamate (64) wasprepared by substituting tert-butyl3-(hydroxymethyl)azetidine-1-carboxylate in Scheme 8 withcyclopentylmethanol. ESI+MS: m/z 317 ([M+H]⁺), 1H NMR (500 MHz,d⁶-DMSO): δ 8.60 (s, 1H), 7.52 (s, 1H), 7.34 (d, J=5 Hz, 1H), 7.23-7.12(m, 2H), 7.03 (dt, J=4.5; 1 Hz, 1H), 6.72 (d, J=8 Hz, 1H), 5.11 (s, 2H),3.96 (d, J=7 Hz, 2H), 2.21 (sept, J=7.5 Hz, 1H), 1.8-1.65 (m, 2H),1.65-1.40 (m, 4H), 1.40-1.15 (m, 2H).

2-Cyclopentylethyl (2-amino-5-(thiophen-2-yl)phenyl)carbamate (65) wasprepared by substituting tert-butyl3-(hydroxymethyl)azetidine-1-carboxylate in Scheme 8 withcyclopentylethanol. ESI+MS: m/z 331 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO):δ 8.59 (s, 1H), 7.52 (s, 1H), 7.34 (d, J=5 Hz, 1H), 7.23-7.13 (m, 2H),7.04 (dt, J=4; 1.5 Hz, 1H), 6.72 (d, J=8 Hz, 1H), 5.11 (s, 2H), 4.08 (t,J=6.5 Hz, 2H), 1.87 (t, J=7 Hz, 1H), 1.81-1.70 (m, 2H), 1.70-1.53 (m,4H), 1.53-1.40 (m, 2H), 1.20-1.05 (m, 2H).

Oxetan-3-ylmethyl (2-amino-5-(thiophen-2-yl)phenyl)carbamate (60) wasprepared by substituting tert-butyl3-(hydroxymethyl)azetidine-1-carboxylate in Scheme 8 withoxetan-3-ylmethanol. ESI+MS: m/z 305 ([M+H]⁺), 1H NMR (500 MHz,d⁶-DMSO): δ 8.70 (bs, 1H), 7.51 (bs, 1H), 7.33 (d, J=5.0 Hz, 1H),7.19-7.17 (m, 2H), 7.03 (t, J=5.0 Hz, 1H), 6.71 (d, J=8.0 Hz, 1H), 5.12(s, 2H), 4.67 (t, J=7.0 Hz, 2H), 4.40 (t, J=6.0 Hz, 2H), 4.29 (d, J=7.0Hz, 2H), 3.21 (m, 1H).

Cyclobutylmethyl (2-amino-5-(thiophen-2-yl)phenyl)carbamate (63) wasprepared by substituting tert-butyl3-(hydroxymethyl)azetidine-1-carboxylate in Scheme 8 withcyclobutylmethanol. ESI+MS: m/z 303 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO):δ 8.62 (bs, 1H), 7.52 (bs, 1H), 7.33 (d, J=5.0 Hz, 1H), 7.19-7.16 (m,2H), 7.04-7.02 (m, 1H), 6.71 (d, J=8.0 Hz, 1H), 5.10 (s, 2H), 4.05 (d,J=6.5 Hz, 2H), 2.64-2.58 (m, 1H), 2.03 (m, 2H), 1.91-1.76 (m, 4H).

2,2-Difluoropropyl (2-amino-5-(thiophen-2-yl)phenyl)carbamate (77) wasprepared by substituting tert-butyl3-(hydroxymethyl)azetidine-1-carboxylate in Scheme 8 with2,2-difluoropropan-1-ol. ESI+MS: m/z 313 ([M+H]⁺), 1H NMR (500 MHz,d⁶-DMSO): δ 8.96 (bs, 1H), 7.49 (bs, 1H), 7.35 (d, J=5.0 Hz, 1H),7.22-7.19 (m, 2H), 7.04-7.02 (m, 1H), 6.73 (d, J=8.5 Hz, 1H), 5.15 (s,2H), 4.36 (t, J=13.5 Hz, 2H), 1.69 (t, J=19.0 Hz, 3H).

(Tetrahydro-2H-pyran-2-yl)methyl(2-amino-5-(thiophen-2-yl)phenyl)carbamate (166) was prepared bysubstituting tert-butyl 3-(hydroxymethyl)azetidine-1-carboxylate inScheme 8 with (tetrahydro-2H-pyran-2-yl)methanol. ESI+MS: m/z 333([M+H]+), 1HNMR (500 MHz, d⁶-DMSO): δ 8.73 (bs, 1H), 7.53 (bs, 1H), 7.33(d, J=5.0 Hz, 1H), 7.18-7.16 (m, 2H), 7.03 (t, J=4.5 Hz, 1H), 6.71 (d,J=8.0 Hz, 1H), 5.11 (bs, 2H), 4.03-3.97 (m, 2H), 3.88 (d, J=10.5 Hz,1H), 3.53 (m, 1H), 3.36 (m, 1H), 1.79 (m, 1H), 1.58 (d, J=13 Hz, 1H),1.47 (bs, 3H), 1.26-1.24 (m, 1H).

(Tetrahydro-2H-pyran-3-yl)methyl(2-amino-5-(thiophen-2-yl)phenyl)carbamate (167) was prepared bysubstituting tert-butyl 3-(hydroxymethyl)azetidine-1-carboxylate inScheme 8 with (tetrahydro-2H-pyran-3-yl)methanol. ESI+MS: m/z 333([M+H]+), 1HNMR (500 MHz, d⁶-DMSO): δ 8.62 (bs, 1H), 7.50 (bs, 1H), 7.33(d, J=5.0 Hz, 1H), 7.19-7.17 (m, 2H), 7.04-7.02 (m, 1H), 6.71 (d, J=8.0Hz, 1H), 5.11 (s, 2H), 4.01-3.82 (m, 3H), 3.73-3.72 (m, 1H), 3.34 (m,1H), 3.21 (t, J=9.5 Hz, 1H), 1.88-1.77 (m, 2H), 1.57-1.48 (m, 2H),1.30-1.23 (m, 1H).

(Tetrahydro-2H-pyran-4-yl)methyl(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)carbamate (169) was prepared bysubstituting tert-butyl 3-(hydroxymethyl)azetidine-1-carboxylate inScheme 8 with (tetrahydro-2H-pyran-4-yl)methanol and by substitutingthiophen-2-ylboronic acid in Scheme 7 with (4-fluorophenyl)boronic acid.ESI+MS: m/z 345 ([M+H]+), 1HNMR (500 MHz, d⁶-DMSO): δ 8.61 (bs, 1H),7.52-7.50 (m, 3H), 7.20-7.15 (m, 3H), 6.75 (d, J=8.5 Hz, 1H), 5.04 (s,2H), 3.93-3.84 (m, 4H), 3.29 (t, J=9.0 Hz, 2H), 1.88 (m, 1H), 1.63-1.58(m, 2H), 1.30-1.26 (m, 2H).

(Tetrahydrofuran-3-yl)methyl (2-amino-5-(thiophen-2-yl)phenyl)carbamate(170) was prepared by substituting tert-butyl3-(hydroxymethyl)azetidine-1-carboxylate in Scheme 8 with(tetrahydrofuran-3-yl)methanol. ESI+MS: m/z 319 ([M+H]+), 1HNMR (500MHz, d⁶-DMSO): δ 8.65 (bs, 1H), 7.50 (bs, 1H), 7.33 (d, J=5.0 Hz, 1H),7.19-7.17 (m, 2H), 7.04-7.02 (m, 1H), 6.72 (d, J=8.0 Hz, 1H), 5.12 (s,2H), 4.08-3.95 (m, 2H), 3.76-3.73 (m, 2H), 3.66-3.61 (m, 1H), 3.61-3.48(m, 1H), 2.56-2.53 (m, 1H), 1.99-1.96 (m, 1H), 1.64-1.59 (m, 1H).

Piperidin-4-ylmethyl (4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)carbamate(90) was prepared by substituting tert-butyl3-(hydroxymethyl)azetidine-1-carboxylate in Scheme 8 with tert-butyl4-(hydroxymethyl)piperidine-1-carboxylate and by substitutingthiophen-2-ylboronic acid in Scheme 7 with (4-fluorophenyl) boronicacid. ESI+MS: m/z 344 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 8.62 (s,1H), 7.58-7.46 (m, 3H), 7.24-7.12 (m, 3H), 6.76 (d, J=7.5 Hz, 1H), 5.05(s, 2H), 3.91 (d, J=6.5 Hz, 2H), 3.31 (s, 1H), 2.98 (d, J=11.5 Hz, 2H),2.60-2.30 (m, 2H), 1.80-1.55 9m, 3H), 1.20-1.05 (m, 2H).

Piperidin-2-ylmethyl (2-amino-5-(thiophen-2-yl)phenyl)carbamate (201)was prepared by substituting tert-butyl3-(hydroxymethyl)azetidine-1-carboxylate in Scheme 8 with tert-butyl2-(hydroxymethyl)piperidine-1-carboxylate. ESI+MS: m/z 332 ([M+H]⁺), 1HNMR (500 MHz, d⁶-DMSO): δ 8.60 (bs, 1H), 7.52 (bs, 1H), 7.33 (d, J=5.0Hz, 1H), 7.21-7.16 (m, 2H), 7.04-7.02 (m, 1H), 6.72 (d, J=8.0 Hz, 1H),5.14 (s, 2H), 3.94-3.87 (m, 2H), 2.95 (d, J=11.0 Hz, 1H), 2.73 (bs, 1H),2.53 (s, 1H), 1.74 (bs, 1H), 1.61-1.50 (m, 2H), 1.33-1.23 (m, 2H),1.09-1.05 (m, 1H).

Piperidin-3-ylmethyl (2-amino-5-(thiophen-2-yl)phenyl)carbamate (153)was prepared by substituting tert-butyl3-(hydroxymethyl)azetidine-1-carboxylate in Scheme 8 with tert-butyl3-(hydroxymethyl)piperidine-1-carboxylate. ESI+MS: m/z 362 ([M+H]+),1HNMR (500 MHz, d⁶-DMSO): δ ESI+MS: m/z 332 ([M+H]⁺); 1H NMR (500 MHz,d⁶-DMSO): δ 8.69 (bs, 1H), 7.51 (bs, 1H), 7.34 (d, J=5.0 Hz, 1H),7.19-7.18 (m, 2H), 7.03 (d, J=4.5 Hz, 1H), 6.72 (d, J=8.5 Hz, 1H), 5.15(bs, 2H), 4.03-3.93 (m, 2H), 3.31-3.16 (m, 3H), 2.73-2.50 (m, 2H), 2.06(bs, 1H), 1.78 (d, J=11.0 Hz, 2H), 2.53 (s, 1H), 1.61-1.59 (m, 1H),1.27-1.23 (m, 1H).

cyclohexyl (2-amino-5-(thiophen-2-yl)phenyl)carbamate (83) was preparedby substituting tert-butyl 3-(hydroxymethyl)azetidine-1-carboxylate inScheme 8 with cyclohexanol. ESI+MS: m/z 317 ([M+H]+).

Alternatively, the Nitro Group Reduction can be Carried Out Using Zincand Ammonium Formate as Described in the Scheme Below:

Synthesis of cyclohexylmethyl (2-amino-5-(thiophen-2-yl)phenyl)carbamate(66)

To a stirred solution of cyclohexylmethyl(2-nitro-5-(thiophen-2-yl)phenyl)carbamate (0.09 g, 0.25 mmol, 1.0equiv.) in methanol (5 mL) and THF (5 mL) were added Zn (0.08 mg, 1.25mmol, 5.0 equiv.) and HCOONH₄ (0.13 mmol, 2.0 mmol, 8.0 equiv.) at roomtemperature. The reaction mixture was stirred at room temperature for 4h then filtered through celite and concentrated under reduced pressure.The crude residue was diluted with ethyl acetate, washed with water andbrine. The combined organic layers were dried over sodium sulfate,filtered and concentrated. The crude residue was purified by columnchromatography (silica gel, 30% EtOAc/hexanes) to affordcyclohexylmethyl (2-amino-5-(thiophen-2-yl)phenyl)carbamate (0.08 g,100% yield). ESI+MS: m/z 331 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 8.58(bs, 1H), 7.52 (bs, 1H), 7.33 (d, J=4.5 Hz, 1H), 7.22-7.14 (m, 2H), 7.03(dd, J=3.5, 5.0 Hz, 1H), 6.72 (d, J=8.0 Hz, 1H), 5.11 (s, 2H), 3.89 (d,J=6.5 Hz, 2H), 1.80-1.38 (m, 6H), 1.25-1.03 (m, 3H), 1.03-0.92 (m, 2H).

One skilled in the art will recognize that other compounds describedbelow were prepared in a similar manner to the procedures describedabove.

Oxetan-3-yl (2-amino-5-(thiophen-2-yl)phenyl)carbamate (59) was preparedby substituting tert-butyl 3-(hydroxymethyl)azetidine-1-carboxylate inScheme 8 with oxetan-3-ol. ESI+MS: m/z 291 ([M+H]⁺), 1H NMR (500 MHz,d⁶-DMSO): δ 8.88 (bs, 1H), 7.49 (bs, 1H), 7.32 (d, J=5.0 Hz, 1H),7.22-7.16 (m, 2H), 7.03 (dd, J=3.5, 4.5 Hz, 1H), 6.72 (d, J=8.5 Hz, 1H),5.44-5.38 (m, 1H), 5.16 (s, 2H), 4.08 (t, J=6.5 Hz, 2H), 4.60-4.54 (m,2H).

Cyclopropyylmethyl (2-amino-5-(thiophen-2-yl)phenyl)carbamate (61) wasprepared by substituting tert-butyl3-(hydroxymethyl)azetidine-1-carboxylate in Scheme 8 withcyclopropylmethanol. ESI+MS: m/z 288 ([M+H]⁺), 1H NMR (500 MHz,d⁶-DMSO): δ 8.67 (bs, 1H), 7.54 (bs, 1H), 7.34 (d, J=5.0 Hz, 1H),7.20-7.15 (m, 2H), 7.03 (dd, J=4.0, 5.0 Hz, 1H), 6.71 (d, J=9.0 Hz, 1H),5.12 (s, 2H), 3.91 (d, J=7.0 Hz, 2H), 1.20-1.12 (m, 1H), 0.52-0.50 (m,2H), 0.35-0.28 (m, 2H).

Isobutyl (2-amino-5-(thiophen-2-yl)phenyl)carbamate (79) was prepared bysubstituting tert-butyl 3-(hydroxymethyl)azetidine-1-carboxylate inScheme 8 with 2-methylpropan-1-ol. ESI+ MS: m/z 291 ([M+H]⁺), 1H NMR(500 MHz, d⁶-DMSO): δ 8.60 (bs, 1H), 7.52 (bs, 1H), 7.34 (d, J=5.0 Hz,1H), 7.20-7.15 (m, 2H), 7.03 (dd, J=3.5, 5.0 Hz, 1H), 6.72 (d, J=9.0 Hz,1H), 5.11 (s, 2H), 3.86 (d, J=6.5 Hz, 2H), 1.92 (sept, J=7.0 Hz, 1H),0.93 (d, J=6.5 Hz, 6H).

Piperidin-4-ylmethyl (2-amino-5-(thiophen-2-yl)phenyl)carbamate (89) wasprepared by substituting tert-butyl3-(hydroxymethyl)azetidine-1-carboxylate in Scheme 8 with tert-butyl4-(hydroxymethyl)piperidine-1-carboxylate. ESI+MS: m/z 332 ([M+H]⁺), 1HNMR (500 MHz, d⁶-DMSO): δ 8.59 (bs, 1H), 7.52 (bs, 1H), 7.34 (d, J=5.0Hz, 1H), 7.21-7.16 (m, 2H), 7.04 (t, J=4.0 Hz, 1H), 6.72 (d, J=8 Hz,1H), 5.11 (bs, 2H), 3.90 (d, J=6.5 Hz, 2H), 2.95 (d, J=11.5 Hz, 2H),2.50-2.40 (m, 2H), 1.80-1.65 (m, 1H), 1.63 (d, J=12.5 Hz, 2H), 1.20-1.05(m, 2H).

Piperidin-4-ylmethyl (2-amino-5-(5-chlorothiophen-2-yl)phenyl)carbamate(179) was prepared by substituting tert-butyl3-(hydroxymethyl)azetidine-1-carboxylate in Scheme 8 with tert-butyl4-(hydroxymethyl)piperidine-1-carboxylate and by substitutingthiophen-2-ylboronic acid in Scheme 7 with 5-chloro-thiophen-2-ylboronicacid. ESI+MS: m/z 366 ([M+H]+), 1H NMR (500 MHz, d⁶-DMSO): δ 8.61 (bs,1H), 7.46 (bs, 1H), 7.13-7.11 (m, 1H), 7.05-7.02 (m, 2H), 6.71 (d, J=8.0Hz, 1H), 5.20 (s, 2H), 3.93-3.89 (m, 2H), 2.95 (d, J=11.0 Hz, 2H),2.50-2.44 (m, 2H), 1.71-1.61 (m, 3H), 1.16-1.08 (m, 2H).

Alternatively, Other Substituted Nitroanilines can be Prepared Accordingto the Scheme Described Below:

Synthesis of 5-(cyclopent-1-en-1-yl)-2-nitroaniline

To a solution of 5-bromo-2-nitroaniline (35.0 g, 161 mmol, 1.0 equiv.)in DMF (4 L) was slowly added sodium hydride (4.64 g, 194 mmol, 1.2equiv.) followed by the dropwise addition of a solution of (Boc)₂O (42.2g, 194 mmol, 1.2 equiv.) in DMF (100 mL) at room temperature. Thereaction mixture was stirred for 4 h at room temperature, then quenchedwith water. The product was extracted with ethyl acetate. The combinedorganic layers were washed with water, dried with sodium sulfate andconcentrated under reduced pressure. The crude residue was purified bycolumn chromatography (silica gel, 1% EtOAc/hexanes) to give tert-butyl(5-bromo-2-nitrophenyl)carbamate (26.6 g, 52% yield).

A mixture of tert-butyl (5-bromo-2-nitrophenyl)carbamate (17.0 g, 53.6mmol, 1.0 equiv.), bis(pinacolato)diboron (20.4 g, 80.0 mmol, 1.5equiv.), potassium acetate (174 g, 1.8 mol, 33 equiv.) and Pd(PPh₃)₄(61.9 g, 53.6 mmol, 1.0 equiv.) in toluene (240 mL) was degassed thenheated to 110° C. After vigorously stirring for 3 h, the solution wasdiluted with water, filtered through celite, washed with ethyl acetate.The oranic layer was separated, dried with magnesium sulfate andconcentrated under reduced pressure. The crude residue was purified byflash column chromatography (silica gel, 20% EtOAc/hexanes) to givetert-butyl(2-nitro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)carbamate(16 g, 82% yield).

To a solution of tert-butyl(2-nitro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)carbamate(16 g, 43.9 mmol, 1.0 equiv.) in 1,4-dioxane (320 mL) were addedcyclopent-1-en-1-yl trifluoromethanesulfonate (11.4 g, 52.7 mmol, 1.2equiv.), Pd (PPh₃)₄ (2.54 g, 2.2 mmol, 0.05 equiv.) and a solution ofNa₂CO₃ (5.6 g, 52.7 mmol, 1.2 equiv.) in water (160 mL) at roomtemperature. The reaction was then heated to 110° C. and stirred for 4h. The reaction mixture was filtered through celite then concentratedunder reduced pressure. The crude residue was purified by columnchromatography (silica gel, 2% EtOAc/hexanes) to give tert-butyl(5-(cyclopent-1-en-1-yl)-2-nitrophenyl)carbamate (4.3 g, 32% yield).

A solution of tert-butyl(5-(cyclopent-1-en-1-yl)-2-nitrophenyl)carbamate (4.3 g, 14.13 mmol, 1.0equiv.) in dichloromethane (50 mL) was added TFA (10 mL) at roomtemperature. The reaction mixture was stirred at room temperature for 2h. The reaction was then evaporated under reduced pressure. The cruderesidue was quenched with a saturated solution of sodium bicarbonate.The product was extracted with ethyl acetate, washed with water andbrine, dried, filtered and concentrated under reduced pressure. Thecrude residue was purified by column chromatography (silica gel, 60%EtOAc/hexanes) to give 5-(cyclopent-1-en-1-yl)-2-nitroaniline (2.5 g,87% yield).

One skilled in the art will recognize that other compounds describedbelow were prepared in a similar manner to the previously describedprocedures using other substituted nitroanilines.

Propyl (2-amino-5-(cyclopent-1-en-1-yl)phenyl)carbamate (82) wasprepared by substituting tert-butyl3-(hydroxymethyl)azetidine-1-carboxylate in Scheme 8 with propanol andby substituting 2-ntro-5-(thiophen-2-yl)aniline in Scheme 8 with5-(cyclopenten-1-yl)nitroaniline. ESI+MS: m/z 261 ([M+H]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 8.51 (bs, 1H), 7.28 (bs, 1H), 7.01 (dd, J=1.5, 8.0 Hz,1H), 6.64 (d, J=8.5 Hz, 1H), 5.91 (s, 1H), 4.93 (s, 2H), 4.00 (t, J=6.5Hz, 2H), 2.56-2.54 (m, 2H), 2.45-2.40 (m, 2H), 1.94-1.90 (m, 2H),1.65-1.60 (m, 2H), 0.93 (t, J=7.5 Hz, 3H).

Cyclopropylmethyl (2-amino-5-(cyclopent-1-en-1-yl)phenyl)carbamate (62)was prepared by substituting tert-butyl3-(hydroxymethyl)azetidine-1-carboxylate in Scheme 8 withcyclopropylmethanol and by substituting 2-ntro-5-(thiophen-2-yl)anilinein Scheme 8 with 5-(cyclopenten-1-yl)nitroaniline. ESI+MS: m/z 273([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 8.58 (bs, 1H), 7.31 (bs, 1H),7.00 (dd, J=2.0, 8.5 Hz, 1H), 6.64 (d, J=8.0 Hz, 1H), 5.91 (s, 1H), 4.94(s, 2H), 3.89 (d, J=7.5 Hz, 2H), 2.58-2.50 (m, 2H), 2.47-2.40 (m, 2H),1.94-1.87 (m, 2H), 1.20-1.08 (m, 1H), 0.56-0.51 (m, 2H), 0.32-0.27 (m,2H).

isobutyl (2-amino-5-(cyclopent-1-en-1-yl)phenyl)carbamate (80) wasprepared by substituting tert-butyl3-(hydroxymethyl)azetidine-1-carboxylate in Scheme 8 with2-methylpropan-1-ol and by substituting 2-ntro-5-(thiophen-2-yl)anilinein Scheme 8 with 5-(cyclopenten-1-yl)nitroaniline. ESI+MS: m/z 275([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 8.51 (bs, 1H), 7.28 (bs, 1H),7.01 (dd, J=1.5, 8.0 Hz, 1H), 6.64 (d, J=8.5 Hz, 1H), 5.91 (s, 1H), 4.93(s, 2H), 3.83 (d, J=6.0 Hz, 2H), 2.56-2.50 (m, 2H), 2.46-2.40 (m, 2H),1.95-1.85 (m, 3H), 0.92 (d, J=6.5 Hz, 6H).

(Tetrahydro-2H-pyran-4-yl) methyl(2-amino-5-(cyclopent-1-en-1-yl)phenyl) carbamate (198) was prepared bysubstituting tert-butyl 3-(hydroxymethyl)azetidine-1-carboxylate inScheme 8 with (tetrahydro-2H-pyran-4-yl)methanol and by substituting2-ntro-5-(thiophen-2-yl)aniline in Scheme 8 with5-(cyclopenten-1-yl)nitroaniline. ESI+MS: m/z 317 ([M+H]+), 1HNMR (500MHz, d⁶-DMSO): δ 8.52 (bs, 1H), 7.27 (bs, 1H), 7.01 (d, J=7.5 Hz, 1H),6.64 (d, J=8.5 Hz, 1H), 5.91 (s, 1H), 4.93 (s, 2H), 3.92-3.84 (m, 5H),2.53-2.42 (m, 5H), 1.92-1.89 (m, 3H), 1.59 (d, J=11.5 Hz, 2H), 1.28-1.25(m, 2H).

Alternatively, Carbamates can be Formed According to the Scheme Below:

Synthesis of ethyl (2-amino-5-(thiophen-2-yl)phenyl)carbamate (81)

To a solution of tert-butyl (2-amino-4-(thiophen-2-yl)phenyl)carbamate(0.20 g, 0.69 mmol, 1.0 equiv.) in dichloromethane (4 mL) was addedethyl chloroformate (0.08 mL, 0.83 mmol, 1.2 equiv.), TEA (0.19 mL, 1.37mmol, 2.0 equiv.) and DMAP (8 mg, 0.07 mmol, 0.1 equiv.) at 0° C. Thereaction was warmed to room temperature and stirred for 16 h. Thereaction was then diluted with dichloromethane and water. The organiclayer was separated, washed with water and brine, dried over sodiumsulfate, filtered and concentrated under reduced pressure. The crudematerial was purified on column chromatography (silica gel, 20%EtOAc/hexanes) to give tert-butyl ethyl(4-(thiophen-2-yl)-1,2-phenylene)dicarbamate (0.11 g, 44% yield).

To a stirred solution of tert-butyl ethyl(4-(thiophen-2-yl)-1,2-phenylene)dicarbamate (0.10 g, 0.28 mmol, 1.0equiv.) in MeOH (2 mL) at 0° C. was added a 4M solution of HCl indioxane (1.5 mL). The reaction was warmed to room temperature andstirred for 2 h, The reaction was concentrated under reduced pressure. Asaturated aqueous solution of sodium bicarbonate was added. The obtainedsolid was filtered, washed with water and dried. The crude material waspurified by column chromatography (silica gel, 15% EtOAc/hexanes) togive ethyl (2-amino-5-(thiophen-2-yl)phenyl)carbamate (0.05 g, 69%yield).

One skilled in the art will recognize that other compounds describedbelow were prepared in a similar manner to the procedures describedabove.

Isopropyl (2-amino-5-(thiophen-2-yl)phenyl)carbamate (78) was preparedby substituting ethyl chloroformate in Scheme 16 with isopropylchloroformate. ESI+MS: m/z 277 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ8.59 (bs, 1H), 7.55 (bs, 1H), 7.34 (dd, J=1.0, 5.0 Hz, 1H), 7.21-7.15(m, 2H), 7.03 (dd, J=4.0, 5.0 Hz, 1H), 6.71 (d, J=8.0 Hz, 1H), 5.12 (s,2H), 4.88 (sept, J=6.0 Hz, 1H), 1.26 (d, J=6.5 Hz, 6H).

Benzyl (2-amino-5-(thiophen-2-yl)phenyl)carbamate (85) was prepared bysubstituting ethyl chloroformate in Scheme 16 with benzyl chloroformate.ESI+MS: m/z 325 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 8.77 (bs, 1H),7.55 (bs, 1H), 7.46-7.32 (m, 6H), 7.22-7.16 (m, 2H), 7.03 (dd, J=4.0,5.0 Hz, 1H), 6.72 (d, J=8.0 Hz, 1H), 5.15 (bs, 2H), 5.14 (bs, 2H).

Synthesis of (1-methylazetidin-3-yl)methyl(2-amino-5-(thiophen-2-yl)phenyl) carbamate (74)

To a solution of tert-butyl3-((((2-nitro-5-(thiophen-2-yl)phenyl)carbamoyl)oxy)methyl)azetidine-1-carboxylate (0.50 g, 1.15 mmol, 1 eq.) in dichloromethane(10 mL) was added TFA (3 mL) at 0° C. The reaction was stirred at roomtemperature for 2 h. The reaction was concentrated under reducedpressure. A saturated solution of sodium bicarbonate was added. Theobtained solid was filtered and dried under reduced pressure to affordazetidin-3-ylmethyl (2-nitro-5-(thiophen-2-yl)phenyl)carbamate (0.35 g,91% yield).

To a stirred solution of azetidin-3-ylmethyl(2-nitro-5-(thiophen-2-yl)phenyl)carbamate (0.09 g, 0.26 mmol, 1.0 eq.)in CH₃CN:CH₂Cl₂ was added aq. formaldehyde (0.77 mmol, 3.0 eq.). Thereaction mixture was stirred at room temperature for 30 min then cooledto 0° C. Sodium cyanoborohydride (0.03 g, 0.46 mmol, 1.8 eq.) was addedslowly. The reaction was quenched with an aqueous saturated solution ofsodium bicarbonate. The product was extracted with ethyl acetate. Thecombined organic layers were washed with brine, dried over sodiumsulfate and concentrated under reduced pressure. The crude product waspurified by column chromatography (silica gel, 5% MeOH/CH₂Cl₂) to obtain(1-methylazetidin-3-yl)methyl (2-nitro-5-(thiophen-2-yl)phenyl)carbamate(0.07 g, 75% yield).

To a stirred solution of (1-methylazetidin-3-yl)methyl(2-nitro-5-(thiophen-2-yl)phenyl)carbamate (0.06 g, 0.16 mmol, 1 eq.) inMeOH (5 mL) was added Pd/C (0.03 g, 0.24 mmol, 1.5 eq). The reaction wasstirred at room temperature under H₂ atmosphere for 2 h. The reactionmixture was filtered through Celite and concentrated. The crude solid,which was washed with ether and pentane to obtain(1-methylazetidin-3-yl)methyl (2-amino-5-(thiophen-2-yl)phenyl)carbamate(0.03 g, 62% yield) as ash color solid. ESI+MS: m/z 318 ([M+H]⁺), 1H NMR(500 MHz, d⁶-DMSO): δ 8.69 (bs, 1H), 7.50 (bs, 1H), 7.34 (d, J=4.5 Hz,1H), 7.22-7.16 (m, 2H), 7.06-7.02 (m, 1H), 6.72 (d, J=8.0 Hz, 1H), 5.13(bs, 2H), 4.19 (d, J=6.5 Hz, 2H), 3.54-3.45 (m, 2H), 3.25-3.15 (m, 2H),2.82-2.70 (m, 1H), 2.35 (s, 3H).

One skilled in the art will recognize that other compounds describedbelow were prepared in a similar manner to the procedures describedabove.

(1-methylpiperidin-4-yl)methyl(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)carbamate (70) was prepared bysubstituting tert-butyl 3-(hydroxymethyl)azetidine-1-carboxylate inScheme 8 with tert-butyl 4-(hydroxymethyl)piperidine-1-carboxylate andby substituting thiophen-2-ylboronic acid in Scheme 2 with(4-fluorophenyl) boronic acid. ESI+MS: m/z 358 ([M+H]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 8.60 (bs, 1H), 7.53-7.59 (m, 3H), 7.21-7.16 (m, 3H),6.76 (d, J=8.0 Hz, 1H), 5.05 (s, 2H), 3.92 (d, J=6.0 Hz, 2H), 2.75 (d,J=11.0 Hz, 2H), 1.82 (t, J=10.5 Hz, 2H), 1.67-1.57 (m, 3H), 1.27-1.21(m, 2H).

(1-Methylpiperidin-3-yl)methyl(2-amino-5-(thiophen-2-yl)phenyl)carbamate (161) was prepared bysubstituting tert-butyl 3-(hydroxymethyl)azetidine-1-carboxylate inScheme 8 with tert-butyl 3-(hydroxymethyl)piperidine-1-carboxylate.ESI+MS: m/z 346 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 8.61 (s, 1H),7.51 (s, 1H), 7.34 (s, 1H), 7.19-7.13 (m, 2H), 7.04-7.02 (m, 1H),6.73-6.68 (m, 1H), 5.12 (s, 2H), 4.03-3.88 (m, 2H), 2.83-2.69 (m, 2H),2.21 (s, 3H), 1.98-1.90 (m, 3H), 1.65 (brs, 2H), 1.49 (d, J=12.0 Hz,1H), 1.02 (d, J=10.0 Hz, 1H).

Alternatively, the nitro group reduction can be carried out using zincand ammonium formate as previously described and one skilled in the artwill recognize that other compounds described below can be prepared in asimilar manner:

(1-Methylpiperidin-4-yl)methyl(2-amino-5-(thiophen-2-yl)phenyl)carbamate (69) was prepared bysubstituting tert-butyl 3-(hydroxymethyl)azetidine-1-carboxylate inScheme 8 with tert-butyl 4-(hydroxymethyl)piperidine-1-carboxylate.ESI+MS: m/z 346 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 8.60 (bs, 1H),7.52 (bs, 1H), 7.33 (d, J=4.5 Hz, 1H), 7.22-7.14 (m, 2H), 7.03 (dd,J=4.0, 5.5 Hz, 1H), 5.11 (s, 2H), 3.93 (d, J=6.5 Hz, 2H), 2.76 (d,J=11.5 Hz, 2H), 2.14 (s, 3H), 1.83 (t, J=11.0 Hz, 2H), 1.66 (d, J=12.5Hz, 2H), 1.63-1.52 (m, 1H), 1.30-1.18 (m, 2H).

(1-Methylpiperidin-4-yl)methyl(2-amino-5-(5-chlorothiophen-2-yl)phenyl)carbamate (197) was prepared bysubstituting tert-butyl 3-(hydroxymethyl)azetidine-1-carboxylate inScheme 8 with tert-butyl 4-(hydroxymethyl)piperidine-1-carboxylate andthiophen-2-ylboronic acid in Scheme 7 with 5-chlorothiophen-2-ylboronicacid. ESI+MS: m/z 380 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 8.62 (bs,1H), 7.46 (bs, 1H), 7.13-7.11 (m, 1H), 7.06-7.03 (m, 2H), 6.71 (d, J=8.0Hz, 1H), 5.21 (bs, 2H), 3.92 (d, J=6.5 Hz, 2H), 2.75 (d, J=11.5 Hz, 2H),2.14 (s, 3H), 1.82 (t, J=11.0 Hz, 2H), 1.66-1.57 (m, 3H), 1.27-1.22 (m,2H).

Synthesis of (1-acetylazetidin-3-yl)methyl(2-amino-5-(thiophen-2-yl)phenyl) carbamate (73)

To a solution of azetidin-3-ylmethyl(2-nitro-5-(thiophen-2-yl)phenyl)carbamate (0.35 g, 1.05 mmol, 1 eq.) indichloromethane (10 mL) was added TEA (0.22 mL, 1.57 mmol, 1.5 eq.) andacetic anhydride (0.11 mL, 1.15 mmol, 1.1 eq.) at 0° C. The reaction waswarmed to room temperature and stirred for 1 h. The reaction was dilutedwith dichloromethane. The organic layer was washed with water and brine.The combined organic layers were dried over sodium sulfate, filtered andconcentrated under reduced pressure. The crude material was purified bycolumn chromatography (silica gel, 4% MeOH/CH₂Cl₂) to obtain(1-acetylazetidin-3-yl)methyl (2-nitro-5-(thiophen-2-yl)phenyl)carbamate(0.31 g, 79% yield).

To a solution of (1-acetylazetidin-3-yl)methyl(2-nitro-5-(thiophen-2-yl)phenyl)carbamate (0.15 g, 0.40 mmol, 1 eq.) inMeOH/THF (5 mL/5 mL) was added Zinc powder (0.13 g, 2.00 mmol, 5 eq.)and ammonium formate (0.20 g, 3.20 mmol, 8 eq.). The reaction wasstirred at room temperature for 3 h. The reaction was filtered throughCelite and the solids washed with MeOH. The filtrate was concentratedunder reduced pressure then diluted with water. The obtained solid wasfiltered and dried. The crude product was purified by columnchromatography (silica gel, 5% MeOH/CH₂Cl₂) to obtain(1-acetylazetidin-3-yl)methyl (2-amino-5-(thiophen-2-yl)phenyl)carbamate(0.030 g, 22% yield). ESI+MS: m/z 346 ([M+H]⁺), 1H NMR (500 MHz,d⁶-DMSO): δ 8.74 (bs, 1H), 7.50 (bs, 1H), 7.34 (d, J=5.0 Hz, 1H),7.24-7.14 (m, 2H), 7.08-7.01 (m, 1H), 6.72 (d, J=8.0 Hz, 1H), 5.14 (bs,2H), 4.30-4.10 (m, 3H), 3.95-3.80 (m, 2H), 3.68-3.55 (m, 1H), 2.95-2.85(m 1H), 1.74 (s, 3H).

One skilled in the art will recognize that other compounds describedbelow can be prepared in a similar manner to the procedures describedabove.

(1-acetylazetidin-3-yl)methyl(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)carbamate (72) was prepared bysubstituting thiophen-2-ylboronic acid in Scheme 7 with (4-fluorophenyl)boronic acid. ESI+MS: m/z 358 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ8.74 (s, 1H), 7.60-7.45 (m, 3H), 7.23-7.15 (m, 3H), 6.77 (d, J=8 Hz,1H), 5.06 (s, 2H), 4.22 (d, J=6.5 Hz, 2H), 4.21-4.12 (m, 1H), 3.94-3.84(m, 2H), 3.58-3.66 (m, 1H), 2.96-2.84 (m, 1H), 1.73 (s, 3H).

(1-Acetylpiperidin-4-yl)methyl(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)carbamate (67) was prepared bysubstituting tert-butyl 3-(hydroxymethyl)azetidine-1-carboxylate inScheme 8 with tert-butyl 4-(hydroxymethyl)piperidine-1-carboxylate andby substituting thiophen-2-ylboronic acid in Scheme 7 with(4-fluorophenyl) boronic acid. ESI+MS: m/z 386 ([M+H]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 8.64 (s, 1H), 7.58-7.46 (m, 3H), 7.24-7.14 (m, 3H),6.77 (d, J=8 Hz, 1H), 5.06 (s, 2H), 4.39 (d, J=12 Hz, 1H), 3.95 (d,J=6.5 Hz, 2H), 3.82 (d, J=14 Hz, 1H), 3.02 (t, J=12.5 Hz, 1H), 1.98 (s,3H), 1.93-1.80 (m, 2H), 1.71 (t, J=14 Hz, 2H), 1.30-1.13 (m, 1H),1.13-1.00 (m, 1H).

(1-Acetylpiperidin-2-yl)methyl(2-amino-5-(thiophen-2-yl)phenyl)carbamate (152) was prepared bysubstituting tert-butyl 3-(hydroxymethyl)azetidine-1-carboxylate inScheme 8 with tert-butyl 2-(hydroxymethyl)piperidine-1-carboxylate.ESI+MS: m/z 374 ([M+H]+), 1HNMR (500 MHz, d⁶-DMSO): δ 8.37 (bs, 1H),7.43 (bs, 1H), 7.28 (d, J=4.5 Hz, 1H), 7.17-7.14 (m, 2H), 7.02-7.01 (m,1H), 6.73 (d, J=8.5 Hz, 1H), 4.90 (s, 2H), 4.48 (s, 1H), 4.16 (m, 1H),2.0 (s, 3H), 1.73 (d, J=7.0 Hz, 1H), 1.64-1.56 (m, 4H), 1.38-1.25 (m,2H).

(1-Acetylpiperidin-3-yl)methyl(2-amino-5-(thiophen-2-yl)phenyl)carbamate (160) was prepared bysubstituting tert-butyl 3-(hydroxymethyl)azetidine-1-carboxylate inScheme 8 with tert-butyl 3-(hydroxymethyl)piperidine-1-carboxylate.ESI+MS: m/z 374 ([M+H]⁺), 1HNMR (500 MHz, d⁶-DMSO): δ 8.66 (brs, 1H),7.50 (brs, 1H), 7.33 (d, J=5.0 Hz, 1H), 7.17 (t, J=3.5 Hz, 2H), 7.03 (d,J=4.0 Hz, 1H), 6.71 (d, J=8.5 Hz, 1H), 5.11 (s, 2H), 4.28 (brs, 1H),4.05-3.87 (m, 2H), 3.69-3.67 (m, 1H), 3.02-2.97 (m, 1H), 2.72 (brs, 1H),1.98 (s, 3H), 1.78-1.59 (m, 3H), 1.42-1.40 (m, 1H), 1.29-1.22 (m, 2H).

(1-Acetylpiperidin-4-yl)methyl(2-amino-5-(5-chlorothiophen-2-yl)phenyl)carbamate (175) was prepared bysubstituting tert-butyl 3-(hydroxymethyl)azetidine-1-carboxylate inScheme 8 with tert-butyl 4-(hydroxymethyl)piperidine-1-carboxylate andby substituting thiophen-2-ylboronic acid in Scheme 7 with(5-chlorothiophen-2-yl)boronic acid. ESI+MS: m/z 408 ([M+H]+), 1H NMR(500 MHz, d⁶-DMSO): δ 8.65 (bs, 1H), 7.46 (bs, 1H), 7.12 (d, J=8.5 Hz,1H), 7.06-7.03 (m, 2H), 6.71 (d, J=8.0 Hz, 1H), 5.22 (s, 2H), 4.38 (d,J=13.0 Hz, 1H), 3.94 (d, J=6.0 Hz, 2H), 3.82 (d, J=13.5 Hz, 1H), 3.01(t, J=12.5 Hz, 1H), 1.98 (s, 3H), 1.88 (m, 1H), 1.71 (t, J=14.5 Hz, 2H),1.28-1.18 (m, 2H), 1.07-1.05 (m, 1H).

Alternatively, the nitro group reduction can be carried out using zincand ammonium formate as previously described and one skilled in the artwill recognize that other compounds described below can be prepared in asimilar manner:

(1-Acetylpiperidin-4-yl)methyl(2-amino-5-(thiophen-2-yl)phenyl)carbamate (68) was prepared bysubstituting tert-butyl 3-(hydroxymethyl)azetidine-1-carboxylate inScheme 8 with tert-butyl 4-(hydroxymethyl)piperidine-1-carboxylate.ESI+MS: m/z 374 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 8.62 (bs, 1H),7.52 (bs, 1H), 7.34 (d, J=4.5 Hz, 1H), 7.21-7.16 (m, 2H), 7.03 (dd,J=3.5, 5.0 Hz, 1H), 6.72 (d, J=8.5 Hz, 1H), 5.12 (s, 2H), 4.39 (d,J=13.0 Hz, 1H), 3.95 (d, J=6.0 Hz, 2H), 3.82 (d, J=13.5 Hz, 1H), 3.02(t, J=13.5 Hz, 1H), 1.99 (s, 3H), 1.95-1.85 (m, 1H), 1.72 (t, J=15.0 Hz,2H), 1.30-1.15 (m, 2H), 1.15-1.02 (m, 1H).

(1-acetylpiperidin-4-yl)methyl (2-aminophenyl)carbamate (240) wasprepared by substituting tert-butyl3-(hydroxymethyl)azetidine-1-carboxylate in Scheme 8 with tert-butyl4-(hydroxymethyl)piperidine-1-carboxylate and2-nitro-5-(thiophen-2-yl)aniline in Scheme 8 with 2-nitroaniline.ESI+MS: m/z 292 ([M+H]⁺); 1H NMR (400 MHz, d6-DMSO): δ 8.51 (bs, 1H),7.17-7.15 (m, 1H), 6.69-6.67 (m, 1H), 6.54-6.50 (m, 1H), 4.84 (s, 2H),4.38 (d, J=12.8 Hz, 1H), 3.91 (d, J=6.4 Hz, 2H), 3.82 (d, J=13.6 Hz,1H), 3.01 (t, J=12.4 Hz, 1H), 2.54-2.50 (m, 1H), 1.98 (s, 3H), 1.87 (bs,1H), 1.70 (t, J=12.8 Hz, 2H), 1.19-1.04 (m, 2H).

Synthesis of 1-(2-amino-5-(thiophen-2-yl)phenyl)-3-isopropylurea (97)

To a solution of tert-butyl (2-amino-4-(thiophen-2-yl)phenyl)carbamate(0.20 g, 0.70 mmol, 1.0 equiv.) in dichloromethane (4 mL) was addedisopropyl isocyanate (0.07 mL, 0.83 mmol, 1.2 equiv.) and TEA (0.19 mL,1.37 mmol) at 0° C. The reaction was stirred at room temperature for 16h. The reaction was then diluted with dichloromethane and water. Theorganic layer was separated, washed with water and brine, dried oversodium sulfate, then concentrated under reduced pressure. The crudematerial was purified by column chromatography (silica gel, 20%EtOAc/hexanes) to give tert-butyl(2-(3-isopropylureido)-4-(thiophen-2-yl)phenyl)carbamate (0.18 g, 70%yield).

A 4M solution of HCl in dioxane (2 mL) was added to a stirred solutionof tert-butyl (2-(3-isopropylureido)-4-(thiophen-2-yl)phenyl)carbamate(0.15 g, 0.40 mmol, 1 equiv.) in methanol (4 mL) at 0° C. The reactionwas warmed to room temperature and stirred for 2 h. The reaction wasthen concentrated under reduced pressure. A saturated aqueous solutionof sodium bicarbonate was added. The obtained solid was filtered, washedwith water and dried to yield1-(2-amino-5-(thiophen-2-yl)phenyl)-3-isopropylurea (0.10 g, 91% yield).ESI+MS: m/z 276 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 7.67 (d, J=2.0Hz, 1H), 7.47 (s, 1H), 7.33 (d, J=5.0 Hz, 1H), 7.17 (d, J=3.0 Hz, 1H),7.09 (dd, J=1.5, 8.0 Hz, 1H), 7.05-7.01 (m, 1H), 6.72 (d, J=8.0 Hz, 1H),6.08 (d, J=8.0 Hz, 1H), 4.87 (s, 2H), 3.76 (sept, J=7.0 Hz, 1H), 1.10(d, J=7.0 Hz, 6H).

One skilled in the art will recognize that other compounds describedbelow can be prepared in a similar manner to the procedures describedabove.

1-(2-Amino-5-(thiophen-2-yl)phenyl)-3-propylurea (96) was prepared bysubstituting isopropyl isocyanate in Scheme 23 with n-propyl isocyanate.ESI+MS: m/z 276 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 7.65 (s, 1H),7.56 (s, 1H), 7.33 (d, J=5.0 Hz, 1H), 7.18 (d, J=3.0 Hz, 1H), 7.10 (dd,J=8.0, 1.5 Hz, 1H), 7.03 (t, J=4.5 Hz, 1H), 6.72 (d, J=8.0 Hz, 1H), 6.21(t, J=5.5 Hz, 1H), 4.90 (s, 2H), 3.05 (t, J=6.5 Hz, 2H), 1.45 (sext,J=6.5 Hz, 2H), 0.89 (t, J=6.5 Hz, 3H).

Synthesis of3-(2-amino-5-(cyclopent-1-en-1-yl)phenyl)-1-methyl-1-propylurea (93)

To a solution of 5-(cyclopent-1-en-1-yl)-2-nitroaniline (0.13 g, 0.61mmol, 1.0 equiv.) in dichloromethane were added triethylamine (0.68 mL,4.90 mmol, 8.0 equiv.) and triphosgene (0.18 g, 0.61 mmol, 1.0 equiv.)at 0° C. The mixture was warmed to room temperature and stirred for 3 hat room temperature. Triethylamine (0.17 mL, 1.22 mmol, 2.0 equiv.) andN-methylpropan-1-amine (0.07 g, 0.92 mmol, 1.5 equiv.) were added slowlyto the reaction mixture. The reaction mixture was slowly warmed to 50°C. and stirred for 2 h. The mixture was diluted with dichloromethane,washed with water and brine, dried over anhydrous magnesium sulfate,filtered, and concentrated under reduced pressure to give crude3-(5-(cyclopent-1-en-1-yl)-2-nitrophenyl)-1-methyl-1-propylurea (0.14 g,76% crude yield) which was used in the next step without furtherpurification.

To a stirred solution3-(5-(cyclopent-1-en-1-yl)-2-nitrophenyl)-1-methyl−1-propylurea (0.17 g,0.56 mmol, 1.0 equiv.) in MeOH were added Zn (0.18 g, 2.80 mmol, 5.0equiv.) and HCOONH₄ (0.28 d, 4.48 mmol, 8.0 equiv.) at room temperature.The reaction was stirred for 2 h then filtered through celite. Theorganic layer was concentrated and the crude residue was purifed bycolumn chromotagraphy (silica gel, 5% MeOH/CH₂Cl₂) to afford3-(2-amino-5-(cyclopent-1-en-1-yl)phenyl)-1-methyl−1-propylurea (0.12 g,78% yield). ESI+MS: m/z 274 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 7.61(s, 1H), 7.08 (d, J=1.5 Hz, 1H), 7.01 (dd, J=8.0, 1.5 Hz, 1H), 6.66 (d,J=8.0 Hz, 1H), 5.94-8.90 (m, 1H), 4.77 (s, 2H), 3.24 (t, J=7.5 Hz, 2H),2.92 (s, 3H), 2.58-2.52 (m, 2H), 2.46-2.40 (m, 2H), 1.95-1.86 (m, 2H),1.52 (sext, J=7.5 Hz, 2H), 0.86 (t, J=7.5 Hz, 3H).

3-(2-Amino-5-(thiophen-2-yl)phenyl)-1-methyl-1-(oxetan-3-yl)urea (95)was prepared by substituting N-methylpropan-1-amine in Scheme 25 withN-methyloxetan-3-amine and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline with2-nitro-5-(thiophen-2-yl)aniline. ESI+MS: m/z 304 ([M+H]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 7.83 (s, 1H), 7.33 (d, J=5.0 Hz, 1H), 7.27 (s, 1H),7.22-7.16 (m, 2H), 7.03 (t, J=4.5 Hz, 1H), 6.73 (d, J=8.5 Hz, 1H), 5.21(quintet, J=7.5 Hz, 1H), 4.98 (bs, 2H), 4.70-4.63 (m, 4H), 3.07 (s, 3H).

1-(2-amino-5-(thiophen-2-yl)phenyl)-3-cyclopropylurea (100) was preparedby substituting N-methylpropan-1-amine in Scheme 25 withcyclopropanamine and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with2-nitro-5-(thiophen-2-yl)aniline. ESI+MS: m/z 273 ([M]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 7.62 (d, J=2.0 Hz, 1H), 7.50 (s, 1H), 7.33 (d, J=4.5Hz, 1H), 7.18 (d, J=3.0 Hz, 1H), 7.11 (dd, J=2.0, 8.0 Hz, 1H), 7.03 (dd,J=3.5, 4.5 Hz, 1H), 6.72 (d, J=8.5 Hz, 1H), 6.47 (s, 1H), 4.90 (s, 2H),2.58-2.50 (m, 1H), 0.65-0.61 (m, 2H), 0.45-0.40 (m, 2H).

1-(2-amino-5-(thiophen-2-yl)phenyl)-3-(oxetan-3-yl)urea was prepared bysubstituting N-methylpropan-1-amine in Scheme 25 with oxetan-3-amine andby substituting 5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with2-nitro-5-(thiophen-2-yl)aniline. ESI+MS: m/z 289 ([M]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 7.67 (s, 1H), 7.59 (s, 1H), 7.33 (d, J=4.5 Hz, 1H),7.18 (d, J=2.5 Hz, 1H), 7.12 (d, J=8.0 Hz, 1H), 7.05-6.96 (m, 2H), 6.73(d, J=8.0 Hz, 1H), 4.93 (bs, 2H), 4.82-4.70 (m, 3H), 4.47-4.43 (m, 2H).

N-(2-amino-5-(5-chlorothiophen-2-yl)phenyl)pyrrolidine-1-carboxamide(117) was prepared by substituting N-methylpropan-1-amine in Scheme 25with pyrrolidine and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with5-(5-chlorothiophen-2-yl)-2-nitroaniline. ESI+MS: m/z 291 ([M]⁺), 1H NMR(300 MHz, d⁶-DMSO): δ 7.49 (s, 1H), 7.28 (d, J=3.0 Hz, 1H), 7.16 (dd,J=3.0, 6.0 Hz, 1H), 7.07-7.02 (m, 2H), 6.73 (d, J=6.0 Hz, 1H), 5.12 (s,2H), 3.45-3.35 (m, 4H), 1.92-1.80 (m, 4H).

N-(2-amino-5-(thiophen-2-yl)phenyl)-3-fluoropyrrolidine-1-carboxamide(122) was prepared by substituting N-methylpropan-1-amine in Scheme 25with 3-fluoropyrrolidine and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with2-nitro-5-(thiophen-2-yl)aniline. ESI+MS: m/z 305 ([M]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 7.62 (s, 1H), 7.35 (d, J=1.5 Hz, 1H), 7.35-7.31 (m,1H), 7.22-7.18 (m, 2H), 7.03 (dd, J=3.5, 5.0 Hz, 1H), 6.74 (d, J=8.0 Hz,1H), 5.44-5.30 (m, 1H), 5.04 (s, 2H), 3.74-3.50 (m, 3H), 3.48-3.40 (m,1H), 2.22-2.00 (m, 2H).

N-(2-amino-5-(thiophen-2-yl)phenyl)-4,4-difluoropiperidine-1-carboxamide(126) was prepared by substituting N-methylpropan-1-amine in Scheme 25with 4,4-difluoropiperidine and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with2-nitro-5-(thiophen-2-yl)aniline. ESI+MS: m/z 338 ([M+H]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 8.09 (s, 1H), 7.33 (d, J=5.0 Hz, 1H), 7.26 (d, J=2.0Hz, 1H), 7.24-7.18 (m, 2H), 7.03 (dd, J=3.5, 5.0 Hz, 1H), 6.73 (d, J=8.0Hz, 1H), 4.98 (s, 2H), 3.57 (t, J=5.5 Hz, 4H), 2.05-1.95 (m, 4H).

N-(2-amino-5-(thiophen-2-yl)phenyl)-3-fluoroazetidine-1-carboxamide(137) was prepared by substituting N-methylpropan-1-amine in Scheme 25with 3-fluoroazetidine and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with2-nitro-5-(thiophen-2-yl)aniline. ESI+MS: m/z 291 ([M]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 7.85 (s, 1H), 7.36-7.32 (m, 2H), 7.22-7.16 (m, 2H),7.05-7.02 (m, 1H), 6.72 (d, J=8.0 Hz, 1H), 5.47-5.30 (m, 1H), 5.07 (s,2H), 4.32-4.22 (m, 2H), 4.05-3.95 (m, 2H).

N-(2-amino-5-(cyclopent-1-en-1-yl)phenyl)morpholine-4-carboxamide (143)was prepared by substituting N-methylpropan-1-amine in Scheme 25 withmorpholine. ESI+MS: m/z 288 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 7.86(s, 1H), 7.07 (d, J=1.5 Hz, 1H), 7.03 (dd, J=1.5, 8.0 Hz, 1H), 6.66 (d,J=8.0 Hz, 1H), 5.92 (bs, 1H), 4.81 (s, 2H), 3.60 (t, J=5 Hz, 4H), 3.40(t, J=5.0 Hz, 4H), 2.58-2.52 (m, 2H), 2.45-2.40 (m, 2H), 1.95-1.88 (m,2H).

N-(4-amino-2′,3′,4′,5′-tetrahydro-[1,1′-biphenyl]-3-yl)pyrrolidine-1-carboxamide(108) was prepared by substituting N-methylpropan-1-amine in Scheme 25with pyrrolidine and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with4-nitro-2′,3′,4′,5′-tetrahydro-[1,1′-biphenyl]-3-amine. ESI+MS: m/z 286([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 7.39 (s, 1H), 7.11 (d, J=2.0 Hz,1H), 6.93 (dd, J=2.5, 8.0 Hz, 1H), 6.64 (d, J=8.5 Hz, 1H), 5.91 (bs,1H), 4.77 (s, 2H), 3.37-3.32 (m, 4H), 2.30-2.24 (m, 2H), 2.16-2.10 (m,2H), 1.88-1.82 (m, 4H), 1.72-1.66 (m, 2H), 1.60-1.54 (m, 2H).

4-acetyl-N-(2-amino-5-(cyclopent-1-en-1-yl)phenyl)piperazine-1-carboxamide(132) was prepared by substituting N-methylpropan-1-amine in Scheme 25with 1-(piperazin-1-yl)ethanone. ESI+MS: m/z 329 ([M+H]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 7.92 (s, 1H), 7.07 (s, 1H), 7.03 (d, J=8.5 Hz, 1H),6.66 (d, J=8.5 Hz, 1H), 5.92 (bs, 1H), 4.82 (s, 2H), 3.50-3.44 (m, 6H),3.44-3.36 (m, 2H), 2.58-2.52 (m, 2H), 2.46-2.40 (m, 2H), 2.03 (s, 3H),1.96-1.86 (m, 2H).

N-(2-amino-5-(cyclopent-1-en-1-yl)phenyl)pyrrolidine-1-carboxamide (119)was prepared by substituting N-methylpropan-1-amine in Scheme 25 withpyrrolidine. ESI+MS: m/z 272 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 7.44(s, 1H), 7.13 (d, J=1.5 Hz, 1H), 7.01 (dd, J=1.5, 8.0 Hz, 1H), 6.66 (d,J=8.0 Hz, 1H), 5.92 (bs, 1H), 4.87 (s, 2H), 3.40-3.30 (m, 4H), 2.60-2.50(m, 2H), 2.46-2.40 (m, 2H), 1.95-1.86 (m, 2H), 1.86-1.60 (m, 4H).

Alternatively, the nitro group reduction can be carried out using Pd/Cand hydrogen as previously described and one skilled in the art willrecognize that other compounds described below can be prepared in asimilar manner:

3-(2-amino-5-(thiophen-2-yl)phenyl)-1-methyl-1-propylurea (94) wasprepared by substituting 5-(cyclopent-1-en-1-yl)-2-nitroaniline inScheme 25 with 2-nitro-5-(thiophen-2-yl)aniline. ESI+MS: m/z 290([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 7.69 (s, 1H), 7.34 (s, 1H), 7.29(bs, 1H), 7.22-7.16 (m, 2H), 7.04 (t, J=5.0, 1H), 6.74 (d, J=8.5 Hz,1H), 4.94 (s, 2H), 3.26 (t, J=7.5 Hz, 2H), 2.94 (s, 3H), 1.53 (sext,J=7.5 Hz, 2H), 0.86 (t, J=7.5 Hz, 3H).

1-(2-Amino-5-(thiophen-2-yl)phenyl)-3-(cyclopropylmethyl)urea (98) wasprepared by substituting N-methylpropan-1-amine in Scheme 25 withcyclopropylmethanamine and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with2-nitro-5-(thiophen-2-yl)aniline. ESI+MS: m/z 288 ([M+H]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 7.66 (d, J=2.0 Hz, 1H), 7.61 (s, 1H), 7.33 (d, J=4.0Hz, 1H), 7.17 (d, J=2.5 Hz, 1H), 7.10 (dd, J=2.0, 8.0 Hz, 1H), 7.03 (dd,J=3.5, 4.5 Hz, 1H), 6.72 (d, J=8.5 Hz, 1H), 6.28 (t, J=5.5 Hz, 1H), 4.90(s, 2H), 2.98 (t, J=5.5 Hz, 2H), 1.00-0.90 (m, 1H), 0.45-0.40 (m, 2H),0.21-0.16 (m, 2H).

N-(2-Amino-5-(thiophen-2-yl)phenyl)pyrrolidine-1-carboxamide (106) wasprepared by substituting N-methylpropan-1-amine in Scheme 25 withpyrrolidine and by substituting 5-(cyclopent-1-en-1-yl)-2-nitroanilinein Scheme 25 with 2-nitro-5-(thiophen-2-yl)aniline. ESI+ MS: m/z 288([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 7.48 (s, 1H), 7.38-7.30 (m, 2H),7.22-7.16 (m, 2H), 7.03 (dd, J=4.0, 5.0 Hz, 1H), 6.73 (d, J=7.5 Hz, 1H),5.01 (s, 2H), 3.38 (t, J=6.0 Hz, 4H), 1.86 (t, J=6.0 Hz, 4H).

N-(2-amino-5-(5-methylthiophen-2-yl)phenyl)pyrrolidine-1-carboxamide(115) was prepared by substituting N-methylpropan-1-amine in Scheme 25with pyrrolidine and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with2-nitro-5-(5-methylthiophen-2-yl)aniline. ESI+MS: m/z 302 ([M+H]⁺), 1HNMR (300 MHz, d⁶-DMSO): δ 7.50 (s, 1H), 7.29 (d, J=3.0 Hz, 1H), 7.11(dd, J=3.0, 9.0 Hz, 1H), 6.97 (d, J=3.0 Hz, 1H), 6.74-6.70 (m, 2H), 4.99(s, 2H), 3.50-3.30 (m, 4H), 2.42 (s, 3H), 1.90-1.80 (m, 4H).

N-(2-amino-5-(4-methylthiophen-2-yl)phenyl)pyrrolidine-1-carboxamide(116) was prepared by substituting N-methylpropan-1-amine in Scheme 25with pyrrolidine and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with2-nitro-5-(4-methylthiophen-2-yl)aniline. ESI+MS: m/z 302 ([M+H]⁺), 1HNMR (300 MHz, d⁶-DMSO): δ 7.49 (s, 1H), 7.35-7.31 (m, 1H), 7.16 (dd,J=2.0, 9.0 Hz, 1H), 7.06-7.02 (m, 1H), 6.94-6.90 (m, 1H), 6.73 (d, J=9.0Hz, 1H), 5.02 (s, 2H), 3.45-3.35 (m, 4H), 2.20 (s, 3H), 1.90-1.80 (m,4H).

N-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)pyrrolidine-1-carboxamide(118) was prepared by substituting N-methylpropan-1-amine in Scheme 25with pyrrolidine and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with4′-fluoro-4-nitro-[1,1′-biphenyl]-3-amine. ESI+MS: m/z 300 ([M+H]⁺), 1HNMR (500 MHz, d⁶-DMSO): δ 7.58-7.50 (m, 2H), 7.48 (s, 1H), 7.38 (d,J=2.5 Hz, 1H), 7.24-7.14 (m, 3H), 6.78 (d, J=8.5 Hz, 1H), 4.97 (s, 2H),3.38 (t, J=6.5 Hz, 4H), 1.86 (t, J=6.5 Hz, 4H).

N-(2-amino-5-(pyridin-4-yl)phenyl)pyrrolidine-1-carboxamide (103) wasprepared by substituting N-methylpropan-1-amine in Scheme 25 withpyrrolidine and by substituting 5-(cyclopent-1-en-1-yl)-2-nitroanilinein Scheme 25 with 2-nitro-5-(pyridin-4-yl)aniline. ESI+MS: m/z 283([M+H]+), 1HNMR (500 MHz, d6-DMSO): δ 8.49 (d, J=6.5 Hz, 2H), 7.55-7.53(m, 3H), 7.50 (s, 1H), 7.38 (dd, J=2.0 Hz, J=8.0 Hz, 1H), 6.81 (d, J=8.0Hz, 1H), 5.23 (s, 2H), 3.39-3.37 (m, 4H), 1.86 (m, 4H).

N-(2-amino-5-(thiophen-2-yl)phenyl)-3,3-difluoropiperidine-1-carboxamide(125) was prepared by substituting N-methylpropan-1-amine in Scheme 25with 3,3-difluoropiperidine and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with2-nitro-5-(thiophen-2-yl)aniline. ESI+MS: m/z 338 ([M+H]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 8.10 (s, 1H), 7.33 (d, J=4.5 Hz, 1H), 7.26-7.18 (m,3H), 7.03 (dd, J=4.0, 5.0 Hz, 1H), 6.74 (d, J=8.5 Hz, 1H), 4.93 (s, 2H),3.78 (t, J=12.0 Hz, 2H), 3.49 (t, J=4.5 Hz, 2H), 2.11-2.00 (m, 2H),1.75-1.66 (m, 2H).

4-Acetyl-N-(2-amino-5-(thiophen-2-yl)phenyl)piperazine-1-carboxamide(129) was prepared by substituting N-methylpropan-1-amine in Scheme 25with 1-(piperazin-1-yl)ethanone and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with2-nitro-5-(thiophen-2-yl)aniline. ESI+MS: m/z 345 ([M+H]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 7.98 (s, 1H), 7.33 (d, J=5.0 Hz, 1H), 7.28 (d, J=1.5Hz, 1H), 7.22-7.18 (m, 2H), 7.04 (t, J=4.0 Hz, 1H), 6.74 (d, J=8.5 Hz,1H), 4.99 (s, 2H), 3.52-3.38 (m, 8H), 2.04 (s, 3H).

N-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)morpholine-4-carboxamide (144)was prepared by substituting N-methylpropan-1-amine with morpholine inScheme 25 and by substituting 5-(cyclopent-1-en-1-yl)-2-nitroaniline inScheme 25 with 4′-fluoro-4-nitro-[1,1′-biphenyl]-3-amine. ESI+MS: m/z316 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 7.92 (s, 1H), 7.58-7.50 (m,2H), 7.31 (d, J=2 Hz, 1H), 7.24-7.16 (m, 3H), 6.78 (d, J=8.5 Hz, 1H),4.93 (s, 2H), 3.62 (t, J=5 Hz, 4H), 3.42 (t, J=5 Hz, 4H).

N-(2-Amino-5-(thiophen-2-yl)phenyl)piperidine-1-carboxamide (124) wasprepared by substituting N-methylpropan-1-amine in Scheme 25 withpiperidine and by substituting 5-(cyclopent-1-en-1-yl)-2-nitroaniline inScheme 25 with 2-nitro-5-(thiophen-2-yl)aniline. ESI+MS: m/z 302([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 7.87 (s, 1H), 7.33 (d, J=4.5 Hz,1H), 7.27 (d, J=2.0 Hz, 1H), 7.22-7.16 (m, 2H), 7.03 (dd, J=3.5, 5.0 Hz,1H), 6.73 (d, J=8.0 Hz, 1H), 4.92 (s, 2H), 3.41 (t, J=5.5 Hz, 4H),1.62-1.54 (m, 2H), 1.54-1.47 (m, 4H).

N-(2-amino-5-(thiophen-2-yl)phenyl)-4-methylpiperazine-1-carboxamide(130) was prepared by substituting N-methylpropan-1-amine in Scheme 25with 1-methylpiperazine and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with2-nitro-5-(thiophen-2-yl)aniline. ESI+MS: m/z 317 ([M+H]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 7.91 (s, 1H), 7.33 (d, J=4.5 Hz, 1H), 7.30-7.25 (m,1H), 7.23-7.16 (m, 2H), 7.04 (dd, J=3.5, 4.5 Hz, 1H), 6.73 (d, J=8.5 Hz,1H), 4.95 (s, 2H), 3.46-3.40 (m, 4H), 2.38-2.28 (m, 4H), 2.21 (s, 3H).

N-(2-amino-5-(thiophen-2-yl)phenyl)azetidine-1-carboxamide (133) wasprepared by substituting N-methylpropan-1-amine in Scheme 25 withazetidine and by substituting 5-(cyclopent-1-en-1-yl)-2-nitroaniline inScheme 25 with 2-nitro-5-(thiophen-2-yl)aniline. ESI+MS: m/z 274([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 7.63 (s, 1H), 7.36-7.28 (m, 2H),7.20-7.16 (m, 2H), 7.03 (dd, J=3.5, 5.0 Hz, 1H), 6.72 (d, J=8.0 Hz, 1H),5.03 (s, 2H), 3.94 (t, J=7.0 Hz, 4H), 2.18 (quintet, J=7.0 Hz, 2H).

(S)—N-(2-amino-5-(thiophen-2-yl)phenyl)-2-methylpyrrolidine-1-carboxamide(123) was prepared by substituting N-methylpropan-1-amine in Scheme 25with (S)-2-methylpyrrolidine and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with2-nitro-5-(thiophen-2-yl)aniline. ESI+MS: m/z 302 ([M+H]⁺), 1H NMR (300MHz, d⁶-DMSO): δ 7.49 (s, 1H), 7.38-7.32 (m, 2H), 7.24-7.16 (m, 2H),7.06-7.02 (m, 1H), 6.74 (d, J=6.0 Hz, 1H), 5.00 (s, 2H), 4.10-3.95 (m,1H), 3.55-3.42 (m, 1H), 2.02-1.80 (m, 4H), 1.60-1.50 (m, 1H), 1.15 (d,J=9.0 Hz, 3H).

N-(2-amino-5-(thiophen-2-yl)phenyl)-8-oxa-3-azabicyclo[3.2.1]octane-3-carboxamide(157) was prepared by substituting N-methylpropan-1-amine in Scheme 25with 8-oxa-3-azabicyclo[3.2.1]octane and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with2-nitro-5-(thiophen-2-yl)aniline. ESI+MS: m/z 330 ([M+H]⁺); 1H NMR (500MHz, d⁶-DMSO): δ 7.79 (bs, 1H), 7.33 (d, J=5.0 Hz, 1H), 7.29 (bs, 1H),7.20-7.18 (m, 2H), 7.03 (t, J=5.0 Hz, 1H), 6.73 (d, J=8.5 Hz, 1H), 4.95(s, 2H), 4.33 (bs, 2H), 3.69 (d, J=13.0 Hz, 2H), 3.03 (d, J=11.0 Hz,2H), 1.82-1.76 (m, 4H).

4-acetamido-N-(2-amino-5-(thiophen-2-yl)phenyl)piperidine-1-carboxamide(113) was prepared by substituting N-methylpropan-1-amine in Scheme 25with N-(piperidin-4-yl)acetamide and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with2-nitro-5-(thiophen-2-yl)aniline. ESI+MS: m/z 359 ([M+H]⁺), 1H NMR (300MHz, d⁶-DMSO): δ 7.98 (s, 1H), 7.86 (d, J=9.0 Hz, 1H), 7.35 (dd, J=1.0,6.0 Hz, 1H), 7.27 (d, J=3.0 Hz, 1H), 7.23-7.18 (m, 2H), 7.05 (dd, J=3.0,4.0 Hz, 1H), 6.75 (d, J=9.0 Hz, 1H), 4.95 (s, 2H), 4.08-3.95 (m, 2H),3.85-3.70 (m, 1H), 3.00-2.86 (m, 2H), 1.81 (s, 3H), 1.81-1.70 (m, 2H),1.40-1.20 (m, 2H).

N-(2-amino-5-cyclopentylphenyl)-3-fluoroazetidine-1-carboxamide (138)was prepared by substituting N-methylpropan-1-amine in Scheme 25 with3-fluoroazetidine and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with5-cyclopentyl-2-nitroaniline. ESI+MS: m/z 278 ([M]⁺), 1H NMR (500 MHz,d⁶-DMSO): δ 7.78 (s, 1H), 6.90 (d, J=2.0 Hz, 1H), 6.75 (dd, J=2.0, 8.0Hz, 1H), 6.61 (d, J=8.0 Hz, 1H), 5.46-5.28 (m, 1H), 4.63 (s, 2H),4.27-4.18 (m, 2H), 3.99-3.91 (m, 2H), 2.83-2.75 (m, 1H), 1.96-1.88 (m,2H), 1.75-1.66 (m, 2H), 1.65-1.54 (m, 2H), 1.49-1.38 (m, 2H).

N-(2-amino-5-(thiophen-2-yl)phenyl)morpholine-4-carboxamide (128) wasprepared by substituting N-methylpropan-1-amine in Scheme 25 withmorpholine and by substituting 5-(cyclopent-1-en-1-yl)-2-nitroaniline inScheme 25 with 2-nitro-5-(thiophen-2-yl)aniline. ESI+MS: m/z 304([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 7.92 (s, 1H), 7.33 (d, J=4.5 Hz,1H), 7.29 (d, J=2.0 Hz, 1H), 7.22-7.18 (m, 2H), 7.03 (dd, J=3.5, 5.0 Hz,1H), 6.73 (d, J=8.0 Hz, 1H), 4.98 (s, 2H), 3.62 (t, J=5.0 Hz, 4H), 3.42(t, J=5.0 Hz, 4H).

N-(2-amino-5-cyclopentylphenyl)-4,4-difluoropiperidine-1-carboxamide(127) was prepared by substituting N-methylpropan-1-amine in Scheme 25with 4,4-difluoropiperidine and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with5-cyclopentyl-2-nitroaniline. ESI+MS: m/z 324 ([M+H]⁺), 1H NMR (500 MHz,d⁶-DMSO): δ 8.03 (s, 1H), 6.84 (d, J=1.5 Hz, 1H), 6.77 (dd, J=1.5, 8.0Hz, 1H), 6.63 (d, J=8.0 Hz, 1H), 4.53 (s, 2H), 3.54 (t, J=5.5 Hz, 4H),2.82-2.75 (m, 1H), 2.02-1.88 (m, 6H), 1.75-1.68 (m, 2H), 1.65-1.55 (m,2H), 1.50-1.38 (m, 2H).

tert-Butyl(1-((2-amino-5-(thiophen-2-yl)phenyl)carbamoyl)pyrrolidin-3-yl)carbamate(114) was prepared by substituting N-methylpropan-1-amine in Scheme 25with tert-butyl pyrrolidin-3-ylcarbamate and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with2-nitro-5-(thiophen-2-yl)aniline. ESI+MS: m/z 403 ([M+H]+), 1H NMR (500MHz, d⁶-DMSO): δ 7.53 (s, 1H), 7.33-7.29 (m, 2H), 7.19-7.17 (m, 3H),7.03-7.02 (m, 1H), 6.72 (d, J=8 Hz, 1H), 5.02 (s, 1H), 4.01-3.95 (m,1H), 3.63-3.56 (m, 1H), 3.54-3.43 (m, 1H), 3.42-3.35 (m, 1H), 2.23-3.16(m, 1H), 2.09-1.97 (m, 1H), 1.86-1.75 (m, 1H), 1.40 (s, 9H).

4-Acetyl-N-(2-amino-5-(4-methylthiophen-2-yl)phenyl)piperazine-1-carboxamide(131) was prepared by substituting N-methylpropan-1-amine in Scheme 25with 1-(piperazin-1-yl)ethanone and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with5-(4-methylthiophen-2-yl)-2-nitroaniline. ESI+MS: m/z 359 ([M+H]+), 1HNMR (500 MHz, d⁶-DMSO): δ 7.99 (s, 1H), 7.25 (d, J=1 Hz, 1H), 7.18 (dd,J=9 Hz, 1 Hz, 1H), 7.04 (s, 1H), 6.91 (s, 1H), 6.73 (d, J=9 Hz, 1H),5.00 (s, 2H), 3.49 (s, 8H), 2.20 (s, 3H), 2.05 (s, 3H).

N-(2-amino-5-(thiophen-2-yl)phenyl)-1-benzylhydrazinecarboxamide (145)was prepared by substituting N-methylpropan-1-amine in Scheme 25 withbenzylhydrazine and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with2-nitro-5-(thiophen-2-yl)aniline. ESI+MS: m/z 339 ([M+H]⁺), 1H NMR (500MHz, d⁶-DMSO): 8.57 (s, 1H), 7.51 (d, J=2.0 Hz, 1H), 7.40-7.26 (m, 6H),7.20 (d, J=3.0 Hz, 1H), 7.14 (dd, J=2.0, 8.0 Hz, 1H), 7.04 (dd, J=8.0;5.0 Hz, 1H), 6.78 (d, J=8.0 Hz, 1H), 4.93 (bs, 2H), 4.67 (bs, 2H), 4.57(bs, 2H).

N-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)-1-benzylhydrazinecarboxamide(154) was prepared by substituting N-methylpropan-1-amine in Scheme 25with benzylhydrazine and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with4′-fluoro-4-nitro-[1,1′-biphenyl]-3-amine. ESI+MS: m/z 351 ([M+H]⁺).1HNMR (500 MHz, d⁶-DMSO): δ 8.57 (s, 1H), 7.73 (d, J=1 Hz, 1H),7.56-7.53 (m, 2H) 7.38-7.35 (m, 2H), 7.31-7.27 (m, 3H), 7.20 (t, J=9.0Hz, 2H), 7.13 (dd, J=2.0 Hz, 1H), 6.82 (d, J=8.5 Hz, 1H), 4.88 (bs, 2H),4.66 (s, 2H), 4.57 (s, 2H).

N-(2-amino-5-(thiophen-2-yl)phenyl)-2-oxa-6-azaspiro[3.3]heptane-6-carboxamide(177) was prepared by substituting N-methylpropan-1-amine in Scheme 25with 2-oxa-6-azaspiro[3.3]heptanes and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with2-nitro-5-(thiophen-2-yl)aniline. ESI+MS: m/z 316 ([M+H]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 7.74 (s, 1H), 7.33-7.30 (m, 2H), 7.19-7.17 (m, 2H),7.03-7.02 (m, 1H), 6.71 (d, J=8.0 Hz, 1H), 5.04 (s, 2H), 4.68 (s, 4H),4.10-4.06 (m, 4H).

N-(2-amino-5-(thiophen-2-yl)phenyl)-7-azabicyclo[2.2.1]heptane-7-carboxamide(181) was prepared by substituting N-methylpropan-1-amine in Scheme 25with 7-azabicyclo[2.2.1]heptane hydrochloride and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with2-nitro-5-(thiophen-2-yl)aniline. ESI+MS: m/z 314 ([M+H]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 8.13 (s, 1H), 7.34-7.32 (m, 2H), 7.20-7.18 (m, 2H),7.03 (t, J=4.5 Hz, 1H), 6.74 (d, J=8.5 Hz, 1H), 4.91 (s, 2H), 3.33 (s,2H), 1.73 (d, J=7.0 Hz, 4H), 1.42 (d, J=6.5 Hz, 4H).

N-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)-3-oxoazetidine-1-carboxamide(203) was prepared by substituting N-methylpropan-1-amine in Scheme 25with azetidin-3-one and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with4′-fluoro-4-nitro-[1,1′-biphenyl]-3-amine. ESI+MS: m/z 300 ([M+H]⁺),¹HNMR (500 MHz, d⁶-DMSO): δ 8.13 (s, 1H), 7.54-7.51 (m, 2H), 7.36 (s,1H), 7.21-7.18 (m, 3H), 6.77 (d, J=8.5 Hz, 1H), 5.08 (s, 2H), 4.77 (s,4H).

3-((1H-imidazol-1-yl)methyl)-N-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)azetidine-1-carboxamide(209) was prepared by substituting N-methylpropan-1-amine in Scheme 25with 1-(azetidin-3-ylmethyl)-1H-imidazole and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with4′-fluoro-4-nitro-[1,1′-biphenyl]-3-amine. ESI+MS: m/z 366 ([M+H]⁺).¹HNMR (400 MHz, d⁶-DMSO): δ 7.76 (S, 1H), 7.67 (s, 1H), 7.52-7.48 (m,2H), 7.33 (d, J=2.0 Hz, 1H), 7.23-7.14 (m, 4H), 6.93 (s, 1H), 6.75 (d,J=8.0 Hz, 1H), 4.23 (d, J=7.6, 2H), 2.96 (t, J=8.4 Hz, 2H), 3.71-3.67(m, 2H) 3.00-2.95 (m, 1H).

N-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)hexahydrocyclopenta[c]pyrrole-2(1H)-carboxamide(210) was prepared by substituting N-methylpropan-1-amine in Scheme 25with octahydrocyclopenta[c]pyrrole hydrochloride and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with4′-fluoro-4-nitro-[1,1′-biphenyl]-3-amine. ESI+MS: m/z 340 ([M+H]⁺),¹HNMR (400 MHz, DMSo-d₆): δ 7.55-7.52 (m, 2H), 7.47 (s, 1H), 7.37 (dJ=2.0 Hz, 1H) 7.22-7.16 (m, 3H), 6.78 (d J=8.0 Hz, 1H) 4.98 (s, 2H) 3.60(dd, J 1,2=8.0 Hz, J 1,3=10.4 Hz, 2H), 3.66 (d J=3.6 Hz, 2H), 1.81-1.70(m, 3H), 1.58-1.54 (m, 1H).

N-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-v)isoindoline-2-carboxamide (212)was prepared by substituting N-methylpropan-1-amine in Scheme 25 withisoindoline and by substituting 5-(cyclopent-1-en-1-yl)-2-nitroanilinein Scheme 25 with 4′-fluoro-4-nitro-[1,1′-biphenyl]-3-amine. ESI+MS: m/z348 ([M+H]⁺), ¹HNMR (400 MHz, d₆-DMSO): δ 7.70 (s, 1H), 7.57-7.54 (m,2H), 7.44 (d, J=2.0 Hz, 1H), 7.38-7.35 (m, 2H), 7.33-7.30 (m, 2H),7.23-7.18 (m, 3H), 6.81 (d, J=8.4 Hz, 1H), 5.06 (s, 2H), 4.79 (s, 4H).

N-(2-amino-5-(pyridin-4-yl)phenyl)isoindoline-2-carboxamide (215) wasprepared by substituting N-methylpropan-1-amine in Scheme 25 withisoindoline and by substituting 5-(cyclopent-1-en-1-yl)-2-nitroanilinein Scheme 25 with 5-(pyridin-4-yl)-2-nitroaniline. ESI+MS: m/z 331([M+H]⁺), ¹HNMR (500 MHz, d⁶-DMSO): δ 8.52 (d, J=4.5 Hz, 2H), 7.74 (s,1H), 7.63 (d, J=6.0 Hz, 3H), 7.47 (d, J=8.5 Hz, 1H), 7.365-7.309 (m,4H), 6.84 (d, J=8.5 Hz, 1H), 5.40 (bs, 2H), 4.79 (s, 4H).

N-(2-amino-5-(pyridin-4-yl)phenyl)hexahydrocyclopenta[c]pyrrole-2(1H)-carboxamide(216) was prepared by substituting N-methylpropan-1-amine in Scheme 25with octahydrocyclopenta[c]pyrrole hydrochloride and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with5-(pyridin-4-yl)-2-nitroaniline. ESI+MS: m/z 323 ([M+H]⁺), ¹HNMR (500MHz, d⁶-DMSO): δ 8.48 (d, J=6.0 Hz, 2H), 7.52 (t, J=17.5 Hz, 4H), 7.38(t, J=8.0 Hz, 1H), 6.80 (d, J=5.0 Hz, 1H), 5.21 (s, 2H), 3.17-3.58 (m,2H), 3.16 (dd, J_(1,2)=3.5 Hz, J_(1,3)=10.0 Hz, 2H), 2.65 (s, 2H),1.80-1.72 (m, 3H), 1.58-1.55 (m, 1H), 1.45-1.42 (m, 2H).

N-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)-5H-pyrrolo[3,4-d]pyrimidine-6(7H)-carboxamide(217) was prepared by substituting N-methylpropan-1-amine in Scheme 25with 6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidine hydrochloride and bysubstituting 5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with4′-fluoro-4-nitro-[1,1′-biphenyl]-3-amine. ESI+MS: m/z 350 ([M+H]⁺),¹HNMR (400 MHz, d⁶-DMSO): δ 9.12 (s, 1H), 8.84 (s, 1H), 7.87 (s, 1H),7.55 (q, J=14 Hz, 2H), 7.40 (d, J=2 Hz, 1H), 7.24-7.19 (m, 3H), 6.80 (d,J=8.4 Hz, 1H), 5.09 (s, 2H), 4.83 (d, J=4 Hz, 4H).

N-(2-aminophenyl)isoindoline-2-carboxamide (218) was prepared bysubstituting N-methylpropan-1-amine in Scheme 25 with isoindoline and bysubstituting 5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with2-nitroaniline. ESI+MS: m/z 254 ([M+H]⁺), ¹HNMR (400 MHz, d⁶-DMSO): δ7.61 (s, 1H), 7.37-7.29 (m, 4H), 7.10 (t, J=8 Hz, 1H), 6.90-6.86 (m,1H), 6.72 (dd, J_(1,2)=1.2 Hz, J_(1,3)=8 Hz, 1H), 6.57-6.53 (m, 1H),4.85 (s, 2H), 4.76 (s, 4H), 4.85 (s, 4H).

N-(2-aminophenyl)-5H-pyrrolo[3,4-b]pyridine-6(7H)-carboxamide (224) wasprepared by substituting N-methylpropan-1-amine in Scheme 25 with6,7-dihydro-5H-pyrrolo[3,4-b]pyridine hydrochloride and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with 2-nitroaniline.ESI+MS: m/z 255 ([M+H]⁺), 1HNMR (300 MHz, MeOD): δ 8.45 (d, J=3.0 Hz,1H), 7.82 (d, J=6.0 Hz, 1H), 7.36 (dd, J=9.0 Hz, J=6.0 Hz, 1H), 7.10 (d,J=9.0 Hz, 1H), 7.0 (t, J=9.0 Hz, 1H), 6.84 (d, J=9.0 Hz, 1H), 6.72 (t,J=9.0 Hz, 1H), 5.49 (s, 1H), 4.83 (br s, 4H).

N-(2-amino-5-(pyridin-4-yl)phenyl)-5-(trifluoromethyl)isoindoline-2-carboxamide(226) was prepared by substituting N-methylpropan-1-amine in Scheme 25with 5-(trifluoromethyl)isoindoline hydrochloride and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with5-(pyridin-4-yl)-2-nitroaniline. ESI+MS: m/z 399 ([M+H]⁺), ¹HNMR (400MHz, d⁶-DMSO): δ 8.50 (d, J=6.0 Hz, 1H), 7.82 (br s, 1H), 7.77 (br s,1H), 7.69-7.67 (m, 1H), 7.61-7.54 (m, 4H), 7.43 (dd, J_(1,2)=2.0 Hz,J_(1,3)=8.4 Hz, 1H), 6.83 (d, J=8.4 Hz, 1H), 5.34 (s, 2H), 4.85 (s, 4H).

N-(2-aminophenyl)-4-methoxyisoindoline-2-carboxamide (227) was preparedby substituting N-methylpropan-1-amine in Scheme 25 with4-methoxyisoindoline hydrochloride and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with 2-nitroaniline.ESI+MS: m/z 284 ([M+H]⁺), ¹HNMR (300 MHz, d⁶-DMSO): δ 7.63 (s, 1H), 7.30(t, J=9.0 Hz, 1H), 7.10 (d, J=9.0 Hz, 1H), 6.96-6.83 (m, 3H), 6.71 (d,J=9.0 Hz, 1H), 6.54 (t, J=9.0 Hz, 1H), 4.86 (br s, 2H), 4.73 (br s, 2H),4.68 (br s, 2H), 3.82 (s, 3H).

N-(2-aminophenyl)-6,7-dihydro-1H-imidazo[4,5-c]pyridine-5(4H)-carboxamide(229) was prepared by substituting N-methylpropan-1-amine in Scheme 25with 4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with 2-nitroaniline.ESI+MS: m/z 258 ([M+H]⁺), ¹HNMR (300 MHz, MeOD): δ 7.52 (s, 1H),7.00-6.80 (m, 2H), 6.79 (d, J=9.0 Hz, 1H), 6.70 (t, J=9.0 Hz, 1H), 4.52(s, 2H), 3.80 (t, J=6.0 Hz, 2H), 2.71 (t, J=6.0 Hz, 2H).

N-(2-aminophenyl)-4-fluoroisoindoline-2-carboxamide (230) was preparedby substituting N-methylpropan-1-amine in Scheme 25 with4-fluoroisoindoline and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with 2-nitroaniline.ESI+MS: m/z 272 ([M+H]⁺), ¹HNMR (300 MHz, d⁶-DMSO): δ 7.72 (s, 1H),7.45-7.35 (m, 1H), 7.25-7.05 (m, 3H), 6.90 (t, J 1 5=9.0 Hz, 1H), 6.72(d, J=9.0 Hz, 1H), 6.55 (t, J=9.0 Hz, 1H), 4.89 (s, 2H), 4.82 (br s,2H), 4.80 (br s, 2H).

N-(2-aminophenyl)-5-(trifluoromethyl)isoindoline-2-carboxamide (246) wasprepared by substituting N-methylpropan-1-amine in Scheme 25 with5-(trifluoromethyl) isoindoline hydrochloride and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with 2-nitroaniline.ESI+MS: m/z 322 ([M+H]⁺), ¹HNMR (300 MHz, MeOD): δ 7.68 (s, 1H),7.65-7.50 (m, 2H), 7.10 (d, J=9.0 Hz, 1H), 7.05-6.97 (m, 1H), 6.85 (d,J=9.0 Hz, 1H), 6.75-6.67 (m, 1H), 4.89 (s, 4H).

N-(2-amino-5-(pyridin-4-yl)phenyl)-4-fluoroisoindoline-2-carboxamide(232) was prepared by substituting N-methylpropan-1-amine in Scheme 25with 4-fluoroisoindoline and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with5-(pyridin-4-yl)-2-nitroaniline. ESI+MS: m/z 349 ([M+H]⁺), ¹HNMR (400MHz, d⁶-DMSO): δ 8.49 (d, J=4.8 Hz, 1H), 7.81 (s, 1H), 7.60-7.50 (m,3H), 7.45-7.35 (m, 2H), 7.22 (d, J=5.6 Hz, 1H), 7.14 (t, J=5.6 Hz, 1H),6.82 (d, J=6.8 Hz, 1H), 5.34 (s, 2H), 4.86-4.78 (m, 4H).

N-(2-amino-5-(pyridin-4-yl)phenyl)-H-pyrrolo[3,4-b]pyridine-6(7H)-carboxamide(233) was prepared by substituting N-methylpropan-1-amine in Scheme 25with 6,7-dihydro-5H-pyrrolo[3,4-b]pyridine and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with5-(pyridin-4-yl)-2-nitroaniline. ESI+MS: m/z 332 ([M+H]⁺), ¹HNMR (400MHz, d⁶-DMSO): δ 8.51-8.48 (m, 3H), 7.82 (d, J=7.6 Hz, 2H), 7.60-7.55(m, 3H), 7.44 (dd, J_(1,2)=2.4 Hz, J_(1,3)=8.4 Hz, 1H), 7.35-7.32 (m,1H), 6.83 (d, J=8.4 Hz, 1H), 5.36 (s, 2H), 4.80 (s, 4H).

N-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)-5H-pyrrolo[3,4-b]pyridine-6(7H)-carboxamide(245) was prepared by substituting N-methylpropan-1-amine in Scheme 25with 6,7-dihydro-5H-pyrrolo[3,4-b]pyridine and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with4′-fluoro-4-nitro-[1,1′-biphenyl]-3-amine. ESI+MS: m/z 349 ([M+H]⁺),¹HNMR (400 MHz, d⁶-DMSO): δ 8.48-8.47 (m, 1H), 7.81-7.79 (m, 2H),7.57-7.53 (m, 2H), 7.41 (d, J=2.0 Hz, 1H), 7.34-7.31 (m, 1H), 7.23-7.18(m, 3H), 6.80 (d, J=8.4 Hz, 1H), 5.07 (s, 2H), 4.79 (s, 4H).

N1-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)-N3,N3-dimethylazetidine-1,3-dicarboxamide(236) was prepared by substituting N-methylpropan-1-amine in Scheme 25with N,N-dimethylazetidine-3-carboxamide and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with4′-fluoro-4-nitro-[1,1′-biphenyl]-3-amine. ESI+MS: m/z 357 ([M+H]⁺),¹HNMR (400 MHz, d⁶-DMSO): δ 7.73 (s, 1H), 7.54-7.51 (m, 2H), 7.36 (d,J=2.4 Hz, 1H), 7.21-7.16 (m, 3H), 6.77 (d, J=8.0 Hz, 1H), 4.99 (s, 2H),4.11-4.01 (m, 4H), 3.72-3.64 (m, 1H), 2.86 (s, 3H), 2.85 (s, 3H).

N-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)-5,6-dihydroimidazo[1,5-a]pyrazine-7(8H)-carboxamide(241) was prepared by substituting N-methylpropan-1-amine in Scheme 25with 5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with4′-fluoro-4-nitro-[1,1′-biphenyl]-3-amine. ESI+MS: m/z 352 ([M+H]⁺),¹HNMR (400 MHz, d⁶-DMSO): δ 8.07 (br s, 1H), 7.59 (br s, 1H), 7.54-7.51(m, 2H), 7.29 (br s, 1H), 7.19-7.16 (m, 3H), 6.76 (t, J=16.4, 2H), 4.96(br s, 2H), 4.70 (br s, 2H), 4.10-4.09 (m, 2H), 3.83-3.82 (m, 2H).

N-(2-amino-5-(pyridin-2-yl)phenyl)pyrrolidine-1-carboxamide (238) wasprepared by substituting N-methylpropan-1-amine in Scheme 25 withpyrrolidine and by substituting 5-(cyclopent-1-en-1-yl)-2-nitroanilinein Scheme 25 with 5-(pyridin-2-yl)-2-nitroaniline. ESI+MS: m/z 283([M+H]⁺), ¹HNMR (400 MHz, d⁶-DMSO): δ 8.52 (d, J=4.4 Hz, 1H), 7.83 (d,J=2.0 Hz, 1H), 7.72-7.71 (m, 2H), 7.63 (dd, J_(1,2)=2.0 Hz, J_(1,3)=8.4Hz, 1H), 7.52 (s, 1H), 7.18-7.15 (m, 1H), 6.77 (d, J=8.4 Hz, 1H), 5.13(s, 2H), 3.38 (t, J=6.4 Hz, 4H), 1.86 (t, J=6.4 Hz, 4H).

N-(2-amino-5-(thiophen-2-yl)phenyl)-2-oxa-6-azaspiro[3.3]heptane-6-carboxamide(177) was prepared by substituting N-methylpropan-1-amine in Scheme 25with 2-oxa-6-azaspiro[3.3]heptane and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with2-nitro-5-(thiophen-2-yl)aniline. ESI+MS: m/z 316 ([M+H]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 9.35 (s, 1H), 8.43 (d, J=2 Hz, 1H), 8.09 (d, J=9 Hz,1H), 7.75 (d, J=5.5 Hz, 1H), 7.68 (d, J=3.5 Hz, 1H), 7.52 (dd, J=2, 8.5Hz, 1H), 4.70 (bs, 4H), 4.22 (bs, 4H).

6-Acetyl-N-(2-amino-5-(thiophen-2-yl)phenyl)-2,6-diazaspiro[3.3]heptane-2-carboxamide(178) can be prepared by substituting N-methylpropan-1-amine in Scheme25 with tert-butyl 2,6-diazaspiro[3.3]heptane-2-carboxylate and bysubstituting 5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with2-nitro-5-(thiophen-2-yl)aniline. ESI+MS: m/z 357 ([M+H]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 7.76 (s, 1H), 7.33-7.31 (m, 2H), 7.19-7.17 (m, 2H),7.03 (dd, J=3.5 Hz, J=5.0 Hz, 1H), 6.71 (d, J=8.5 Hz, 1H), 5.05 (s, 2H),4.25 (s, 2H), 4.07 (s, 4H), 3.97 (s, 2H), 1.73 (s, 3H).

N-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)-2-oxa-6-azaspiro[3.3]heptane-6-carboxamide(186) can be prepared by substituting N-methylpropan-1-amine in Scheme25 with 2-oxa-6-azaspiro[3.3]heptanes and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with4′-fluoro-4-nitro-[1,1′-biphenyl]-3-amine. ESI+MS: m/z 328 ([M+H]⁺), 1HNMR (500 MHz, d⁶-DMSO): δ 7.73 (s, 1H), 7.51 (dd, I=5.5 Hz, I=8.5 Hz,2H), 7.32 (d, J=1.5 Hz, 1H), 7.21-7.16 (m, 3H), 6.76 (d, J=8.5 Hz, 1H),4.98 (s, 2H), 4.68 (s, 4H), 4.10 (s, 4H).

6-Acetyl-N-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)-2,6-diazaspiro[3.3]heptane-2-carboxamide(187) was prepared by substituting N-methylpropan-1-amine in Scheme 25with 1-(2,6-diazaspiro[3.3]heptan-2-yl)ethanone and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with4′-fluoro-4-nitro-[1,1′-biphenyl]-3-amine. ESI+MS: m/z 369 ([M+H]⁺), 1HNMR (500 MHz, d⁶-DMSO): δ 7.76 (s, 1H), 7.53-7.50 (m, 2H), 7.33 (d,J=1.5 Hz, 1H), 7.21-7.17 (m, 3H), 6.76 (d, J=8.0 Hz, 1H), 4.99 (s, 2H),4.25 (s, 2H), 4.07 (s, 4H), 3.97 (s, 2H), 1.73 (s, 3H).

N-(2-amino-4-fluorophenyl)pyrrolidine-1-carboxamide (222) was preparedby substituting N-methylpropan-1-amine in Scheme 25 with pyrrolidine andby substituting 5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with4-fluoro-2-nitroaniline. ESI+MS: m/z 254 ([M+H]⁺), 1H NMR (00 MHz,d6-DMSO): δ 7.37 (bs, 1H), 6.95 (dd, J₁=6 Hz, J₂=9 Hz, 1H), 6.47 (dd,J₁=3 Hz, J₂=12 Hz, 1H), 6.28 (dt, J₁=3 Hz, J₂=9 Hz, 1H), 5.09 (s, 2H),3.35-3.31 (m, 4H) m, 1.90-1.81 (m, 4H).

N-(2-amino-4-fluorophenyl)hexahydrocyclopenta[c]pyrrole-2(1H)-carboxamide(220) was prepared by substituting N-methylpropan-1-amine in Scheme 25with 3-azabicyclo[3,3,0]octane hydrochloride and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with4-fluoro-2-nitroaniline. ESI+MS: m/z 264 ([M+H]⁺), 1HNMR (300 MHz,CDCl₃): δ 7.00-6.93 (m, 1H), 6.50-6.30 (m, 2H), 5.95 (s, 1H), 3.70-3.50(m, 2H), 3.20-3.10 (m, 2H), 2.80-2.60 (br s, 2H), 1.90-1.70 (m, 3H),1.70-1.55 (m, 1H), 1.55-1.40 (m, 2H).

N-(2-amino-5-(pyridin-4-yl)phenyl)-3-azabicyclo[3.1.0]hexane-3-carboxamide(247) was prepared by substituting N-methylpropan-1-amine in Scheme 25with 6,7-dihydro-5H-pyrrolo[3,4-b]pyridine and by substituting5-(cyclopent-1-en-1-yl)-2-nitroaniline in Scheme 25 with5-(pyridin-4-yl)-2-nitroaniline. ESI+MS: m/z 295 ([M+H]+); 1H NMR (400MHz, d6-DMSO): δ 8.50-8.48 (m, 2H), 7.54-7.51 (m, 4H), 7.38 (dd, J=2.4Hz, J=8.4 Hz), 6.80 (d, J=8.4, 1H), 5.21 (s, 2H), 3.61 (d, J=10.4 Hz),3.41-3.35 (m, 2H), 1.58 (t, J=7.6 Hz, 2H), 0.72-0.67 (m, 1H), 0.20-0.16(m, 1H).

Synthesis of1-((1-acetylpiperidin-4-yl)methyl)-3-(2-amino-5-(thiophen-2-yl)phenyl)urea(156)

To a solution of 2-nitro-5-(thiophen-2-yl)aniline (0.60 g, 2.72 mmol,1.0 equiv.) in dichloromethane at 0° C. were added TEA (5 mL, 35.4 mmol,13 equiv.) and triphosgene (0.81 g, 2.72 mmol, 1.0 equiv.). The mixturewas warmed to room temperature and stirred 3 h. The reaction was thencooled to 0° C. and tert-butyl 4-(aminomethyl)piperidine-1-carboxylate(0.88 g, 4.09 mmol, 1.5 equiv.) was then added slowly. The reactionmixture was slowly warmed to room temperature and stirred for 2 h. Thereaction was diluted with dichloromethane, washed with water and brine,dried over anhydrous magnesium sulfate, filtered, and concentrated. Thecrude residue was purified by column chromatography (silica gel,EtOAc/hexanes) to afford tert-butyl4-((3-(2-nitro-5-(thiophen-2-yl)phenyl)ureido)methyl)piperidine-1-carboxylate(0.31 g, 25% yield).

To a solution of tert-butyl4-((3-(2-nitro-5-(thiophen-2-yl)phenyl)ureido)methyl)piperidine-1-carboxylate(0.30 g, 0.65 mmol) in dichloromethane (15 mL) was added TFA (3 mL) at0° C. The reaction was warmed to room temperature and stirred for 2 h.The reaction was concentrated. The crude residue was diluted with asaturated aqueous solution of sodium bicarbonate. The obtained solid wasfiltered, washed with water then washed with ether and pentane to afford1-(2-nitro-5-(thiophen-2-yl)phenyl)-3-(piperidin-4-ylmethyl)urea (0.20g, 85% yield).

Acetic anhydride (0.03 g, 0.31 mmol, 1.1 equiv.) was added to a solutionof 1-(2-nitro-5-(thiophen-2-yl)phenyl)-3-(piperidin-4-ylmethyl)urea(0.10 g, 0.28 mmol, 1.0 equiv.) and triethylamine (60 μL, 0.42 mmol, 1.5equiv.) in dichloromethane (5 mL) at 0° C. The reaction mixture waswarmed to room temperature and stirred for 2 h. The reaction was thendiluted with water. The product was extracted with dichloromethane,washed with water and brine, dried over sodium sulfate, filtered andconcentrated under reduced pressure. The crude solid was purified bycolumn chromatography (silica gel, 5% MeOH/CH₂Cl₂) to afford1-((1-acetylpiperidin-4-yl)methyl)-3-(2-nitro-5-(thiophen-2-yl)phenyl)urea(0.09 g, 81% yield).

To a solution of1-((1-acetylpiperidin-4-yl)methyl)-3-(2-nitro-5-(thiophen-2-yl)phenyl)urea(0.09 g, 0.22 mmol) in methanol was added Pd/C (0.03 g, 0.028 mmol, 1.2equiv.). The reaction mixture was degassed then stirred at roomtemperature under H₂ atmosphere for 1 h. The reaction was filteredthrough celite and concentrated. The crude solid was washed with ether,and dried under reduced pressure to obtain of pure1-((1-acetylpiperidin-4-yl)methyl)-3-(2-amino-5-(thiophen-2-yl)phenyl)urea(0.05 g, 60% yield). ESI+MS: m/z 373 ([M]⁺). 1H NMR (500 MHz, d⁶-DMSO):δ 7.66 (d, J=2.0 Hz, 1H), 7.58 (s, 1H), 7.33 (d, J=5.0 Hz, 1H), 7.17 (d,J=3.5 Hz, 1H), 7.10 (dd, J=3.5 Hz, 1H), 7.04-7.02 (m, 1H), 6.72 (d,J=8.0 Hz, 1H), 6.31 (t, J=6.0 Hz, 1H), 4.89 (bs, 2H), 4.36 (d, J=13.0Hz, 1H), 3.81 (d, J=13.5 Hz, 1H), 3.0-2.96 (m, 3H), 1.90 (s, 3H),1.71-1.64 (m, 3H), 1.10-1.07 (m, 1H), 0.98-0.95 (m, 1H).

One skilled in the art will recognize that other compounds describedbelow can be prepared in a similar manner:

1-((1-acetylazetidin-3-yl)methyl)-3-(2-amino-5-(thiophen-2-1)phenyl)urea(105) was prepared by substituting tert-butyl4-(aminomethyl)piperidine-1-carboxylate in Scheme 27 with tert-butyl3-(aminomethyl)azetidine-1-carboxylate. ESI+MS: m/z 345 ([M+H]⁺), 1H NMR(500 MHz, d⁶-DMSO): δ 7.68-7.60 (m, 2H), 7.36-7.30 (m, 1H), 7.22-7.00(m, 3H), 6.75-6.70 (m, 1H), 6.48-6.42 (m, 1H), 4.92 (bs, 2H), 4.15-4.02(m, 1H), 3.90-3.70 (m, 2H), 3.60-3.50 (m, 1H), 3.40-3.20 (m, 2H),2.80-2.60 (m, 1H), 1.73 (s, 3H).

3-amino-N-(2-amino-5-(thiophen-2-yl)phenyl)pyrrolidine-1-carboxamide(109) was prepared by substituting tert-butyl4-(aminomethyl)piperidine-1-carboxylate in Scheme 27 with tert-butylpyrrolidin-3-ylcarbamate. ESI+MS: m/z 303 ([M+H]⁺).

(R)-3-acetamido-N-(2-amino-5-(thiophen-2-yl)phenyl)pyrrolidine-1-carboxamide(110) was prepared by substituting tert-butyl4-(aminomethyl)piperidine-1-carboxylate in Scheme 27 with (R)-tert-butylpyrrolidin-3-ylcarbamate. ESI+MS: m/z 345 ([M+H]⁺), 1H NMR (500 MHz,d⁶-DMSO): δ 8.13 (d, J=6.5 Hz, 1H), 7.55 (bs, 1H), 7.37-7.32 (m, 2H),7.22-7.16 (m, 2H), 7.03 (dd, J=3.5, 5.0 Hz, 1H), 6.73 (d, J=8.0 Hz, 1H),5.02 (s, 2H), 4.28-4.20 (m, 1H), 3.62-3.58 (m, 1H), 3.58-3.40 (m, 2H),3.23-3.20 (m, 1H), 2.09-2.02 (m, 1H), 1.83 (s, 3H), 1.83-1.75 (m, 1H).

(S)-3-acetamido-N-(2-amino-5-(thiophen-2-yl)phenyl)pyrrolidine-1-carboxamide(111) was prepared by substituting tert-butyl4-(aminomethyl)piperidine-1-carboxylate in Scheme 27 with (S)-tert-butylpyrrolidin-3-ylcarbamate. ESI+MS: m/z 345 ([M+H]⁺), 1H NMR (500 MHz,d⁶-DMSO): δ 8.13 (d, J=6.5 Hz, 1H), 7.55 (bs, 1H), 7.37-7.32 (m, 2H),7.22-7.16 (m, 2H), 7.03 (dd, J=3.5, 5.0 Hz, 1H), 6.73 (d, J=8.0 Hz, 1H),5.02 (s, 2H), 4.28-4.20 (m, 1H), 3.62-3.58 (m, 1H), 3.58-3.40 (m, 2H),3.23-3.20 (m, 1H), 2.09-2.02 (m, 1H), 1.83 (s, 3H), 1.83-1.75 (m, 1H).

3-(acetamidomethyl)-N-(2-amino-5-(thiophen-2-yl)phenyl)azetidine-1-carboxamide(139) was prepared by substituting tert-butyl4-(aminomethyl)piperidine-1-carboxylate in Scheme 27 with tert-butyl(azetidin-3-ylmethyl)carbamate. ESI+MS: m/z 345 ([M+H]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 8.01 (bs, 1H), 7.65 (bs, 1H), 7.38-7.30 (m, 2H),7.20-7.14 (m, 2H), 7.06-7.00 (m, 1H), 6.72 (d, J=8.5 Hz, 1H), 5.04 (s,2H), 3.96 (t, J=8.5 Hz, 2H), 3.63 (t, J=5.5 Hz, 2H), 3.30-3.20 (m, 2H),2.68-2.60 (m, 1H), 1.82 (s, 3H).

6-acetamido-N-(2-amino-5-(thiophen-2-yl)phenyl)-3-azabicyclo[3.1.0]hexane-3-carboxamide(141) was prepared by substituting tert-butyl4-(aminomethyl)piperidine-1-carboxylate in Scheme 27 with tert-butyl3-azabicyclo[3.1.0]hexan-6-ylcarbamate. ESI+MS: m/z 357 ([M+H]⁺), 1H NMR(500 MHz, d⁶-DMSO): δ 7.99 (s, 1H), 7.51 (s, 1H), 7.35-7.30 (m, 2H),7.22-7.16 (m, 2H), 7.03 (dd, J=4.0 Hz, 1H), 6.72 (d, J=8.0 Hz, 1H), 5.00(s, 2H), 3.66 (d, J=10.0 Hz, 2H), 3.43 (d, J=10.0 Hz, 2H), 2.42-2.35 (m,1H), 1.78 (s, 3H), 2.20-2.14 (m, 2H).

1-((1-Acetylazetidin-3-yl)methyl)-3-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)urea(162) was prepared by substituting tert-butyl4-(aminomethyl)piperidine-1-carboxylate in Scheme 27 with tert-butyl3-(aminomethyl)azetidine-1-carboxylate and2-nitro-5-(thiophen-2-yl)aniline in Scheme 27 with4′-fluoro-4-nitro-[1,1′-biphenyl]-3-amine. ESI+MS: m/z 357 ([M+H]⁺); 1HNMR (500 MHz, d⁶-DMSO): δ 7.65-7.60 (m, 2H), 7.56-7.50 (m, 2H), 7.19 (t,J=9.0 Hz, 2H), 7.09 (d, J=6.5 Hz, 1H), 6.76 (d, J=8.5 Hz, 1H), 6.46 (t,J=6.0 Hz, 1H), 4.86 (s, 2H), 4.12 (t, J=8.0 Hz, 1H), 3.85-3.78 (m, 2H),3.56-3.53 (m, 1H), 3.31-3.26 (m, 2H), 2.70-2.67 (m, 1H), 1.72 (s, 3H).

3-acetamido-N-(2-amino-5-cyclopentylphenyl)azetidine-1-carboxamide (135)was prepared by substituting tert-butyl4-(aminomethyl)piperidine-1-carboxylate in Scheme 27 with tert-butylazetidin-3-ylcarbamate and by substituting2-nitro-5-(thiophen-2-yl)aniline with 5-cyclopentyl-2-nitroaniline.ESI+MS: m/z 317 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 8.47 (d, J=7.0Hz, 1H), 7.69 (s, 1H), 6.89 (d, J=1.5 Hz, 1H), 6.75 (dd, J=1.5, 8.5 Hz,1H), 6.62 (d, J=8.5 Hz, 1H), 4.61 (bs, 2H), 4.45-4.35 (m, 1H), 4.12 (t,J=8.0 Hz, 2H), 3.72 (dd, J=5.5, 8.0 Hz, 2H), 1.83-1.74 (m, 1H),1.96-1.86 (m, 2H), 1.83 (s, 3H), 1.78-1.65 (m, 2H), 1.65-1.54 (m, 2H),1.48-1.38 (m, 2H).

1-((1-Acetylazetidin-3-yl)methyl)-3-(2-amino-5-(thiophen-2-yl)phenyl)-1-methylurea(199) was prepared by substituting tert-butyl4-(aminomethyl)piperidine-1-carboxylate in Scheme 27 with tert-butyl3-((methylamino)methyl)azetidine-1-carboxylate. ESI+MS: m/z 359([M+H]⁺); 1H NMR (500 MHz, d⁶-DMSO): δ 7.75 (s, 1H), 7.32 (d, J=5.5 Hz,1H), 7.27 (s, 1H), 7.19 (m, 2H), 7.02 (t, J=4.0 Hz, 1H), 6.73 (d, J=8.0,1H), 4.95 (s, 2H), 4.14 (t, J=8.5 Hz, 1H), 3.88-3.83 (m, 2H), 3.58-3.53(m, 3H), 2.96 (s, 3H), 2.83 (m, 1H), 1.71 (s, 3H).

6-acetyl-N-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)-2,6-diazaspiro[3.4]octane-2-carboxamide(202) was prepared by substituting tert-butyl4-(aminomethyl)piperidine-1-carboxylate in Scheme 27 with tert-butyl2,6-diazaspiro[3.4]octane-6-carboxylate 2-nitro-5-(thiophen-2-yl)anilinein Scheme 27 with 4′-fluoro-4-nitro-[1,1′-biphenyl]-3-amine. ESI+MS: m/z383 ([M+H]⁺), ¹HNMR (500 MHz, CDCl₃): δ 7.73 (d, J=19.5 Hz, 1H),7.54-7.51 (m, 2H), 7.37-7.36 (m, 1H), 7.21-7.17 (m, 3H), 6.77 (dd,J_(1,2)=2.5 Hz, J_(1,3)=8.0 Hz, 1H), 5.01 (s, 2H), 3.93-3.83 (m, 4H),3.61 (s, 1H), 3.47-3.45 (m, 2H), 3.32-3.29 (m, 1H), 2.12 (t, J=7.0 Hz,1H), 2.03 (t, J=7.0 Hz, 1H), 1.93 (d, J=5.5 Hz, 3H).

Alternatively, the nitro group reduction can be carried out using zincand ammonium formate as previously described and one skilled in the artwill recognize that other compounds described below can be prepared in asimilar manner:

3-acetamido-N-(2-amino-5-(thiophen-2-yl)phenyl)azetidine-1-carboxamide(134) was prepared by substituting tert-butyl4-(aminomethyl)piperidine-1-carboxylate in Scheme 27 with tert-butylazetidin-3-ylcarbamate. ESI+MS: m/z 331 ([M+H]⁺), 1H NMR (500 MHz,d⁶-DMSO): δ 8.49 (d, J=6.0 Hz, 1H), 7.77 (s, 1H), 7.36-7.32 (m, 2H),7.22-7.16 (m, 2H), 7.03 (dd, J=3.5, 5.0 Hz, 1H), 6.72 (d, J=8.5 Hz, 1H),5.05 (s, 2H), 4.44-4.40 (m, 1H), 4.17 (t, J=8.0 Hz, 2H), 3.76 (dd,J=6.0, 9.0 Hz, 2H), 1.83 (s, 3H).

3-acetamido-N-(4-amino-2′,3′,4′,5′-tetrahydro-[1,1′-biphenyl]-3-yl)azetidine-1-carboxamide(136) was prepared by tert-butyl 4-(aminomethyl)piperidine-1-carboxylatein Scheme 27 with tert-butyl azetidin-3-ylcarbamate and by substituting2-nitro-5-(thiophen-2-yl)aniline with 5-(1-cyclohexenyl)-2-nitroaniline.ESI+MS: m/z 329 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 8.48 (d, J=6.5Hz, 1H), 7.68 (s, 1H), 7.08 (d, J=2.0 Hz, 1H), 6.94 (dd, J=1.5, 8.5 Hz,1H), 6.63 (d, J=8.5 Hz, 1H), 5.91 (bs, 1H), 4.80 (s, 2H), 4.45-4.36 (m,1H), 4.13 (t, J=8.0 Hz, 2H), 3.72 (dd, J=6.0, 9.0 Hz, 2H), 2.29-2.22 (m,2H), 2.16-2.10 (m, 2H), 1.83 (s, 3H), 1.72-1.65 (m, 2H), 1.60-1.53 (m,2H).

(S)-3-acetamido-N-(4-amino-2′,3′,4′,5′-tetrahydro-[1,1′-biphenyl]-3-yl)pyrrolidine-1-carboxamide(112) was prepared by substituting tert-butyl4-(aminomethyl)piperidine-1-carboxylate in Scheme 27 with (S)-tert-butylpyrrolidin-3-ylcarbamate and by substituting2-nitro-5-(thiophen-2-yl)aniline with 5-(1-cyclohexenyl)-2-nitroaniline.ESI+MS: m/z 343 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 8.11 (d, J=6.5Hz, 1H), 7.45 (s, 1H), 7.10 (d, J=1.0 Hz, 1H), 6.94 (dd, J=1.0, 8.0 Hz,1H), 6.64 (d, J=8.0 Hz, 1H), 5.91 (bs, 1H), 4.78 (s, 2H), 4.28-4.20 (m,1H), 3.62-3.54 (m, 1H), 3.50-3.38 (m, 2H), 3.22-3.16 (m, 1H), 2.30-2.24(m, 2H), 2.16-2.10 (m, 2H), 2.10-2.00 (m, 1H), 1.82 (s, 3H), 1.82-1.74(m, 1H), 1.72-1.64 (m, 2H), 1.60-1.54 (m, 2H).

3-(acetamidomethyl)-N-(4-amino-2′,3′,4′,5′-tetrahydro-[1,1′-biphenyl]-3-yl)azetidine-1-carboxamide(140) was prepared by substituting tert-butyl4-(aminomethyl)piperidine-1-carboxylate in Scheme 27 with tert-butyl(azetidin-3-ylmethyl)carbamate and by substituting2-nitro-5-(thiophen-2-yl)aniline with 5-(1-cyclohexenyl)-2-nitroaniline.ESI+MS: m/z 343 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 8.00 (t, J=5.5Hz, 1H), 7.56 (s, 1H), 7.09 (d, J=2.0 Hz, 1H), 6.93 (dd, J=2.0, 8.0 Hz,1H), 6.62 (d, J=8.0 Hz, 1H), 5.91 (bs, 1H), 4.79 (s, 2H), 3.92 (t, J=8.0Hz, 2H), 3.59 (dd, J=5.5, 8.5 Hz, 2H), 3.25 (t, J=6.0 Hz, 2H), 2.65-2.55(m, 1H), 2.29-2.22 (m, 2H), 2.15-2.10 (m, 2H), 1.82 (s, 3H), 1.72-1.65(m, 2H), 1.60-1.53 (m, 2H).

6-acetamido-N-(4-amino-2′,3′,4′,5′-tetrahydro-[1,1′-biphenyl]-3-yl)-3-azabicyclo[3.1.0]hexane-3-carboxamide(142) was prepared by substituting tert-butyl4-(aminomethyl)piperidine-1-carboxylate in Scheme 27 with tert-butyl3-azabicyclo[3.1.0]hexan-6-ylcarbamate and by substituting2-nitro-5-(thiophen-2-yl)aniline with 5-(1-cyclohexenyl)-2-nitroaniline.ESI+MS: m/z 355 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 7.99 (d, J=3.0Hz, 1H), 7.42 (s, 1H), 7.06 (d, J=2.0 Hz, 1H), 6.94 (dd, J=2.0, 8.0 Hz,1H), 6.63 (d, J=8.0 Hz, 1H), 5.91 (bs, 1H), 4.75 (s, 2H), 3.64 (d,J=10.5 Hz, 2H), 3.40 (d, J=10.5 Hz, 2H), 2.38-2.34 (m, 1H), 2.28-2.24(m, 2H), 2.14-2.08 (m, 2H), 1.78 (s, 3H), 1.72-1.64 (m, 4H), 1.60-1.54(m, 2H).

3-(acetamidomethyl)-N-(2-amino-4-fluorophenyl)azetidine-1-carboxamide(228) was prepared by substituting tert-butyl4-(aminomethyl)piperidine-1-carboxylate in Scheme 27 with tert-butyl(azetidin-3-ylmethyl)carbamate and by substituting2-nitro-5-(thiophen-2-yl)aniline with 4-fluoro-2-nitroaniline. ESI+MS:m/z 281 ([M+H]+); 1H NMR (400 MHz, d6-DMSO): δ 7.99 (m, 1H), 7.51 (s,1H), 6.95 (dd, J=6.4 Hz, J=8.8 Hz, 1H), 6.46 (dd, J=2.8 Hz, J=11.2 Hz,1H), 6.29-6.24 (m, 1H), 5.09 (s, 2H), 3.90 (t, J=8.4 Hz, 2H), 3.59-3.55(m, 2H), 3.24 (t, J=6.4 Hz, 2H), 2.62-2.59 (m, 1H), 1.81 (s, 3H).

Synthesis of1-(2-Amino-5-(thiophen-2-yl)phenyl)-3-((1-methylpiperidin-4-yl)methyl)urea(155)

To a solution of1-(2-nitro-5-(thiophen-2-yl)phenyl)-3-(piperidin-4-ylmethyl)urea inacetonitrile (16 mL) and dichloromethane (8 mL) 0° C. was addedformaldehyde. The reaction was warmed to room temperature then stirredfor 1 h. Sodium cyanoborohydride was then added and the reaction wasstirred for an additional hour. The reaction was quenched with asaturated aqueous solution of sodium bicarbonate. The product wasextracted with ethyl acetate. The combined organic layers were washedwith water and brine, dried, filtered and evaporated under reducedpressure. The crude residue was purified by column chromatography(silica gel, 5% MeOH/CH₂Cl₂) to afford1-(2-nitro-5-(thiophen-2-yl)phenyl)-3-(piperidin-4-ylmethyl)urea (0.045g, 43% yield).

To a solution of1-(2-nitro-5-(thiophen-2-yl)phenyl)-3-(piperidin-4-ylmethyl)urea (0.045g, 0.12 mmol, 1.0 equiv.) in methanol was added Pd/C (0.02 g, 0.02 mmol,0.16 equiv.). The reaction mixture was stirred at room temperature underH₂ balloon pressure for 1 h. The reaction was filtered through celiteand concentrated under reduced pressure. The crude solid was washed withether to obtain of pure1-(2-Amino-5-(thiophen-2-yl)phenyl)-3-((1-methylpiperidin-4-yl)methyl)urea(0.03 g, 67% yield). ESI+MS: m/z 345 ([M]⁺). 1H NMR (500 MHz, d⁶-DMSO):δ 7.66 (d, J=1.5 Hz, 1H), 7.56 (s, 1H), 7.33 (d, J=5.0 Hz, 1H), 7.17 (d,J=3.0 Hz, 1H), 7.10 (dd, J=3.5 Hz, 1H), 7.03 (t, J=4.0 Hz, 1H), 6.71 (d,J=7.5 Hz, 1H), 6.27 (t, J=5.5 Hz, 1H), 4.88 (bs, 2H), 2.98 (t, J=5.5 Hz,2H), 2.77 (d, J=11.0 Hz, 2H), 2.16 (s, 3H), 1.85 (m, 2H), 1.63 (d, J=4.5Hz, 2H), 1.36-1.33 (m, 1H), 1.20-1.13 (m, 3H).

Synthesis of1-(2-amino-5-(thiophen-2-yl)phenyl)-3-(piperidin-4-ylmethyl)urea (159)

To a solution of1-(2-nitro-5-(thiophen-2-yl)phenyl)-3-(piperidin-4-ylmethyl)urea (0.10g, 0.28 mmol, 1.0 equiv.) in methanol was added Pd/C (0.03 g, 0.03 mmol,0.10 equiv.). The reaction mixture was stirred at room temperature underH₂ balloon pressure for 1 h. The reaction was filtered through celiteand concentrated under reduced pressure. The crude solid was washed withether to obtain of pure1-(2-amino-5-(thiophen-2-yl)phenyl)-3-(piperidin-4-ylmethyl)urea (0.06g, 65% yield). ESI+MS: m/z 331 ([M]⁺); 1H NMR (500 MHz, d⁶-DMSO): δ 7.64(d, J=12.0 Hz, 2H), 7.33 (d, J=5.5 Hz, 1H), 7.17 (d, J=3.0 Hz, 1H),7.10-7.09 (m, 1H), 7.03 (t, J=5.0 Hz, 1H), 6.71 (d, J=8.0 Hz, 1H), 6.34(m, 1H), 4.90-4.89 (bs, 2H), 3.16 (d, J=12.5 Hz, 2H), 2.99 (t, J=6.0 Hz,3H), 2.65 (t, J=12.5 Hz, 2H), 1.71 (d, J=13.0 Hz, 1H), 1.62-1.60 (m,2H), 1.20-1.13 (m, 2H).

One skilled in the art will recognize that other compounds describedbelow can be prepared in a similar manner:

3-(2-Amino-5-(thiophen-2-yl)phenyl)-1-(azetidin-3-ylmethyl)-1-methylurea(165) was prepared by substituting tert-butyl4-(aminomethyl)piperidine-1-carboxylate in Scheme 27 with tert-butyl3-((methylamino)methyl)azetidine-1-carboxylate. ESI+MS: m/z 317([M+H]⁺); 1H NMR (500 MHz, d⁶-DMSO): δ 7.27 (d, J=4.0 Hz, 1H), 7.15 (s,1H), 7.05-6.96 (m, 2H), 6.88 (s, 1H), 6.62 (d, J=8.0 Hz, 1H), 5.57 (bs,1H), 4.76-4.68 (m, 3H), 3.38 (s, 2H), 3.18-3.16 (m, 1H), 3.03 (t, J=9.0Hz, 1H), 2.97 (s, 3H), 2.82 (t, J=9.5 Hz, 1H), 2.06 (bs, 1H).

N-(2-amino-5-(thiophen-2-yl)phenyl)-2-benzylhydrazinecarboxamide (146)was prepared by substituting tert-butyl4-(aminomethyl)piperidine-1-carboxylate in Scheme 27 withbenzylhydrazine. ESI+MS: m/z 339 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO):7.97 (bs, 1H), 7.60 (bs, 1H), 7.50-7.41 (m, 3H), 7.38-7.24 (m, 4H),7.18-7.10 (m, 2H), 7.06-7.03 (m, 1H), 6.73 (d, J=8.5 Hz, 1H), 5.26 (bs,1H), 4.77 (bs, 2H), 3.90 (d, J=4.5 Hz, 2H).

N-(2-amino-5-(thiophen-2-yl)phenyl)hydrazinecarboxamide (147) wasprepared by substituting tert-butyl4-(aminomethyl)piperidine-1-carboxylate in Scheme 27 with tert-butylhydrazinecarboxylate. ESI+MS: m/z 249 ([M+H]⁺), 1H NMR (500 MHz,d⁶-DMSO): 8.03 (s, 1H), 7.65 (s, 1H), 7.39 (s, 1H), 7.34 (d, J=5.0 Hz,1H), 7.19 (d, J=3.0 Hz, 1H), 7.13 (dd, J=8.5; 1.5 Hz, 1H), 7.04 (t,J=4.5 Hz, 1H), 6.75 (d, J=8.5 Hz, 1H), 4.93 (s, 2H), 4.32 (s, 2H).

N-(2-amino-5-(thiophen-2-yl)phenyl)-1-propylhydrazinecarboxamide (172)was prepared by substituting tert-butyl4-(aminomethyl)piperidine-1-carboxylate in Scheme 27 with tert-butyl1-propylhydrazinecarboxylate. ESI+MS: m/z 291 ([M]⁺). 1H NMR (500 MHz,d⁶-DMSO): δ 8.54 (s, 1H), 7.70 (s, 1H), 7.34 (d, J=5.0 Hz, 1H), 7.19 (d,J=3.0 Hz, 1H), 7.12-7.10 (m, 1H), 7.04-7.03 (m, 1H), 6.76 (d, J=8.0 Hz,1H), 4.88 (s, 2H), 4.66 (s, 2H), 3.39 (t, J=7.0 Hz, 2H), 1.60-1.56 (m,2H), 0.85 (t, J=7.5 Hz, 3H).

N-(2-amino-5-(thiophen-2-yl)phenyl)tetrahydropyridazine-1(2H)-carboxamide(174) was prepared by substituting tert-butyl4-(aminomethyl)piperidine-1-carboxylate in Scheme 27 with tert-butyltetrahydropyridazine-1(2H)-carboxylate. ESI+MS: m/z 303 ([M+H]⁺); 1H NMR(500 MHz, d⁶-DMSO): δ 8.45 (s, 1H), 7.70 (s, 1H), 7.34 (d, J=5.0 Hz,1H), 7.19 (d, J=2.5 Hz, 1H), 7.12 (d, J=8.0, 1H), 7.03 (m, 1H), 6.76 (d,J=8.0 Hz, 1H), 4.86-4.82 (m, 3H), 3.30 (s, 2H), 2.85 (bs, 2H), 1.57 (bs,4H).

N-(2-amino-5-(thiophen-2-yl)phenyl)pyrazolidine-1-carboxamide (185) canbe prepared by substituting tert-butyl4-(aminomethyl)piperidine-1-carboxylate in Scheme 27 with tert-butylpyrazolidine-1-carboxylate. ESI+MS: m/z 289 ([M+H]⁺), 1H NMR (500 MHz,d⁶-DMSO): δ 8.40 (s, 1H), 7.70 (s, 1H), 7.33 (d, J=5.0 Hz, 1H), 7.19 (d,J=3.5 Hz, 1H), 7.13 (d, J=8.0 Hz, 1H), 7.03 (t, J=4.0 Hz, 1H), 6.77 (d,J=8.5 Hz, 1H), 5.08 (m, 1H), 4.88 (s, 2H), 3.38 (t, J=7.5 Hz, 2H), 2.86(q, J=7.0 Hz, 2H), 1.97 (m, 2H).

N-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)pyrazolidine-1-carboxamide(194) was prepared by substituting tert-butyl4-(aminomethyl)piperidine-1-carboxylate in Scheme 27 with tert-butylpyrazolidine-1-carboxylate and by substituting2-nitro-5-(thiophen-2-yl)aniline with4′-fluoro-4-nitro-[1,1′-biphenyl]-3-amine. ESI+MS: m/z 301 ([M+H]⁺).¹HNMR (500 MHz, d⁶-DMSO): δ 8.42 (s, 1H), 7.67 (s, 1H), 7.54-7.52 (m,2H), 7.20 (t, J=9.0 Hz, 2H), 7.11 (d, J=6.5 Hz, 1H), 6.81 (d, J=8.5 Hz,1H), 5.08 (t, J=8.5 Hz, 1H), 4.83 (s, 2H), 3.38 (t, J=7.0 Hz, 2H),2.88-2.83 (m, 2H), 2.00-1.95 (m, 2H).

N-(2-amino-5-(thiophen-2-yl)phenyl)tetrahydropyridazine-1(2H)-carboxamide(193) can be prepared by substituting tert-butyl4-(aminomethyl)piperidine-1-carboxylate in Scheme 27 with tert-butyltetrahydropyridazine-1(2H)-carboxylate and by substituting2-nitro-5-(thiophen-2-yl)aniline with4′-fluoro-4-nitro-[1,1′-biphenyl]-3-amine. ESI+MS: m/z 315 ([M+H]⁺), 1HNMR (500 MHz, d⁶-DMSO): δ 8.46 (s, 1H), 7.68 (s, 1H), 7.55-7.52 (m, 2H),7.20 (t, J=9.0 Hz, 2H), 7.11 (d, J=8.5 Hz, 1H), 6.82 (d, J=8.0 Hz, 1H),4.83 (bs, 2H), 3.49 (bs, 2H), 2.85 (bs, 2H), 1.58 (bs, 4H).

N-(2-amino-5-(pyridin-4-yl)phenyl)tetrahydropyridazine-1(2H)-carboxamide(235) can be prepared by substituting tert-butyl4-(aminomethyl)piperidine-1-carboxylate in Scheme 27 with tert-butyltetrahydropyridazine-1(2H)-carboxylate and by substituting2-nitro-5-(thiophen-2-yl)aniline in Scheme 27 with2-nitro-5-(pyridin-4-yl)aniline. ESI+MS: m/z 298 ([M+H]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 8.50 (d, J=7.0 Hz, 2H), 8.45 (s, 1H), 7.82 (s, 1H),7.53 (d, J=7.5 Hz, 2H), 7.33 (d, J=10.0 Hz, 1H), 6.84 (d, J=10.5 Hz,1H), 5.08 (s, 2H), 4.83 (t, J=9.0 Hz, 1H), 3.51 (bs, 2H), 2.86 (bs, 2H),1.58 (bs, 4H).

Synthesis of the 3-(2-amino-5-(thiophen-2-yl)phenyl)-1,1-dimethylurea(92)

To a solution of tert-butyl 2-amino-4-(thiophen-2yl)phenylcarbamate(0.12 g, 0.41 mmol, 1.0 equiv.) in pyridine (2 mL) was added a solutionof dimethylcarbamic chloride (0.07 g, 0.62 mmol, 1.5 equiv.) in toluene(0.3 mL). The resulting solution was stirred at room temperatureovernight. The crude residue was diluted with water (10 mL) andextracted with ethyl acetate (10 mL×2). The combined organic layers werewashed with water (10 mL), dried, filtered and concentrated. The cruderesidue was washed with petroleum ether (5 mL) to afford tert-butyl(2-(3,3-dimethylureido)-4-(thiophen-2-yl)phenyl)carbamate (0.12 g, 80%yield).

To a solution of tert-butyl(2-(3,3-dimethylureido)-4-(thiophen-2-yl)phenyl)carbamate (115 mg, 0.32mmol) in dichloromethane (5 mL) was added TFA (2 mL) at 0° C. Thereaction was warmed to room temperature and stirred for 1 h. Thereaction was concentrated. The crude residue was diluted with ethylacetate (30 mL) and washed with a saturated aqueous solution of sodiumbicarbonate, then water (20 mL), dried over sodium sulfate, filtered andconcentrated. The obtained residue was washed with hexane (2 mL) toafford 3-(2-amino-5-(thiophen-2-yl)phenyl)-1,1-dimethylurea (65 mg, 83%yield). ESI+MS: m/z 262 ([M]⁺). 1H NMR (500 MHz, d⁶-DMSO): δ 7.68 (s,1H), 7.34 (d, J=5.0, 1H), 7.29 (d, J=2.0, 1H), 7.19 (dd, J=4.5, 2.0,2H), 7.04 (m, 1H), 6.73 (d, J=8.5, 1H), 5.00 (s, 2H), 2.93 (s, 6H).

One skilled in the art will recognize that other compounds describedbelow can be prepared in a similar manner:

N-(2-amino-5-cyclopropylphenyl)pyrrolidine-1-carboxamide (107) wasprepared by substituting dimethylcarbamic chloride in Scheme 34 withpyrrolidine-1-carbonyl chloride and by substituting tert-butyl(2-amino-4-(thiophen-2-yl)phenyl)carbamate in Scheme 34 with tert-butyl(2-amino-4-cyclopropylphenyl)carbamate. ESI+MS: m/z 246 ([M+H]⁺), 1H NMR(500 MHz, d⁶-DMSO): δ 7.39 (s, 1H), 6.78 (s, 1H), 6.60 (s, 2H), 4.59 (s,2H), 3.40-3.37 (m, 2H), 1.86-1.83 (m, 4H), 1.76-1.70 (m, 1H), 0.82-0.77(m, 2H), 0.49-0.45 (m, 2H).

N-(2-amino-5-vinylphenyl)pyrrolidine-1-carboxamide (120) was prepared bysubstituting dimethylcarbamic chloride in Scheme 34 withpyrrolidine-1-carbonyl chloride and by substituting tert-butyl(2-amino-4-(thiophen-2-yl)phenyl)carbamate in Scheme 34 with tert-butyl(2-amino-4-vinylphenyl)carbamate. ESI+MS: m/z 403 ([M+H]⁺), 1H NMR (500MHz, d⁶-DMSO): δ 7.44 (s, 1H), 7.15 (s, 1H), 6.99 (d, J=9 Hz, 1H), 6.66(d, J=8 Hz, 1H), 6.53 (dd, J=11.5 Hz, 6.5 Hz, 1H), 5.46 (d, J=18 hz,1H), 4.98 (s, 2H), 4.94 (d, J=11 Hz, 1H), 3.40-3.35 (m, 4H0, 1.90-1.75(m, 1H).

Synthesis of theN-(2-amino-5-(pyridin-3-yl)phenyl)pyrrolidine-1-carboxamide (121)

To a solution of 5-bromo-2-nitroaniline (1 g, 4.61 mmol) indichloromethane (15 mL) were added TEA (8.35 mL, 59.9 mmol, 1.3 eq.) andtriphosgene (1.37 g, 4.61 mmol, 1 equiv.) at 0° C. The mixture wasstirred for three hours at room temperature and the pyrolidine (0.39 g,5.53 mmol, 1.2 equiv.) was added slowly. The reaction was stirred for anadditional two hours. The reaction crude was diluted withdichloromethane and washed with water and brine. The organic layers weredried over MgSO₄, filtered and evaporated. The residue was purified bycolumn chromatography on eluting ethyl acetate and hexane to allowN-(2-amino-5-bromophenyl)pyrrolidine-1-carboxamide (0.45 g, 31% yield).

To a solution of N-(2-amino-5-bromophenyl)pyrrolidine-1-carboxamide(0.26 g, 0.72 mmol) in THF (10 mL) and water (5 mL) were addedpyridin-3-ylboronic acid (0.13 g, 1.07 mmol, 1.5 equiv.), sodiumbicarbonate (0.11 g, 1.07 mmol, 1.5 equiv.) and palladium(0) (0.08 g,0.07 mmol, 0.1 equiv.). The reaction mass was stirred at 90° C. forsixteen hours. The reaction crude was filtered on a celite bed anddiluted in ethyl acetate. The organic layers were washed with water andbrine then dried over Na₂SO₄ and concentrated. The residue was purifiedby column chromatography on eluting ethyl acetate and hexane to allowN-(2-amino-5-(pyridin-3-yl)phenyl)pyrrolidine-1-carboxamide (0.14 g, 63%yield).

To a solution ofN-(2-amino-5-(pyridin-3-yl)phenyl)pyrrolidine-1-carboxamide (0.07 g,0.22 mmol) in methanol was added Pd/C (0.03 g, 0.24 mmol, 1.05 equiv.).The reaction mixture was degassed then stirred at room temperature underH₂ atmosphere for 1 h. The reaction was filtered through celite andconcentrated. The crude solid was washed with ether, and dried underreduced pressure to obtain of pureN-(2-amino-5-(pyridin-3-yl)phenyl)pyrrolidine-1-carboxamide (0.04 g, 56%yield). ESI+MS: m/z 283 ([M]⁺). 1H NMR (500 MHz, d⁶-DMSO): δ 8.76 (d,J=1.5 Hz, 1H), 8.42 (d, J=3.5 Hz, 1H), 7.90 (d, J=7.5 Hz, 1H), 7.50 (s,1H), 7.44 (d, J=2 Hz, 1H), 7.39 (dd, J=4.5 Hz, 3 Hz, 1H), 7.27 (dd, J=2Hz, 6 Hz, 1H), 6.82 (d, J=8.5 Hz, 1H), 5.07 (s, 2H), 3.39 (m, 4H), 1.87(m, 4H).

Synthesis of theN-(2-amino-5-(pyrazin-2-yl)phenyl)pyrrolidine-1-carboxamide (168)

To a solution of N-(2-amino-5-bromophenyl)pyrrolidine-1-carboxamide (0.5g, 1.59 mmol) in toluene (25 mL) were added4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (0.60 g,2.39 mmol, 1.5 equiv.), potassium acetate (0.46 g, 4.78 mmol, 3.0equiv.) and Pd(PPh₃)₄ (0.19 g, 0.16 mmol, 0.1 equiv.). The mixture washeated to 110° C. After vigorously stirring for four hours, the solutionwas diluted with water, filtered through celite and washed with ethylacetate. The organic layers were dried over Mg2SO4 and concentrated. Theresidue was purified by flash column by eluting ethyl aceate and hexaneto allowN-(2-nitro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)pyrrolidine-1-carboxamide(0.64 g, 40% yield).

To a solution ofN-(2-nitro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)pyrrolidine-1-carboxamide(0.20 g, 0.55 mmol) was added at room temperature sodium bicarbonate(0.09 g, 0.80 mmol, 1.45 equiv.), 2-bromopyrazine (0.14 g, 0.89 mmol,1.6 equiv.) and palladium (0.07 g, 0.06 mmol, 0.1 equiv). The reactionwas refluexed for fourteen hours. The crude reaction was filtered andconcentrated. The residue was diluted into water and washed with ethylacetate, the concentrated. The obtained was purified through columnchromatography by eluting ethyl acetate and hexane to allowN-(2-nitro-5-(pyrazin-2-yl)phenyl)pyrrolidine-1-carboxamide (0.12 g, 70%yield).

To a solution of 1N-(2-nitro-5-(pyrazin-2-yl)phenyl)pyrrolidine-1-carboxamide (0.11 g,0.35 mmol) in methanol (6 mL) and THF (6 mL) was added Pd/C (0.04 g,0.94 mmol, 0.33 equiv.). The reaction mixture was degassed then stirredat room temperature under H₂ atmosphere for 1 h. The reaction wasfiltered through celite and concentrated. The crude solid was washedwith ether, and dried under reduced pressure to obtain of pureN-(2-amino-5-(pyrazin-2-yl)phenyl)pyrrolidine-1-carboxamide (0.07 g, 70%yield). ESI+MS: m/z 284 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 9.30 (s,1H), 8.55 (s, 1H), 8.40-8.39 (m, 1H), 7.88 (s, 1H), 7.71-7.69 (m, 1H),7.53 (s, 1H), 6.81 (d, J=8.5 Hz, 1H), 5.31 (s, 2H), 3.40-3.37 (m, 4H),1.86 (m, 4H).

Synthesis of N1-(2-amino-5-(thiophen-2-yl)phenyl)oxalamide (1)

To a solution of tert-butyl (2-amino-4-(thiophen-2-yl)phenyl)carbamate(1.0 g, 3.44 mmol) in dichloromethane (10 mL) was added ethyl oxalylchloride (0.6 mL, 5.17 mmol, 1.5 equiv.) and TEA (1.2 mL, 8.61 mmol, 2.5equiv.) at 0° C. The reaction was warmed to room temperature and stirredfor 4 h. The reaction was then diluted with dichloromethane and water.The combined organic layers were washed with water and brine, dried overNa₂SO₄ and then concentrated under reduced pressure. The crude materialwas purified by column chromatography (silica gel, EtOAc/hexanes) toafford ethyl2-((2-((tert-butoxycarbonyl)amino)-5-(thiophen-2-yl)phenyl)amino)-2-oxoacetate(1.0 g, 74% yield).

To a solution of ethyl2-((2-((tert-butoxycarbonyl)amino)-5-(thiophen-2-yl)phenyl)amino)-2-oxoacetate(0.2 g, 0.512 mmol) in MeOH (2 mL) was added methanolic ammonia (5 mL,0.512 mmol, 1 equiv.) in a sealed tube. The reaction was stirred at roomtemperature for five h. The obtained white solid was filtered and driedto afford tert-butyl(2-(2-amino-2-oxoacetamido)-4-(thiophen-2-yl)phenyl)carbamate (0.11 g,59% yield).

To a solution of tert-butyl(2-(2-amino-2-oxoacetamido)-4-(thiophen-2-yl)phenyl)carbamate (0.1 g,0.28 mmol) in DCM (3 mL) was added TFA (1 mL, 13 mmol) at 0° C. Thereaction was warmed to room temperature and stirred for 2 h. Thereaction was then concentrated under reduced pressure. The residue wasbasified with a saturated aqueous solution of Na₂CO₃. The obtained solidwas filtered, washed with water and ether then dried to affordN1-(2-amino-5-(thiophen-2-yl)phenyl)oxalamide (40 mg, 55% yield).ESI+MS: m/z 262 ([M+H]⁺), 1H NMR (500 MHz, d⁶-DMSO): δ 9.89 (s, 1H),8.24 (s, 1H), 7.94 (s, 1H), 7.59 (d, J=2.0 Hz, 1H), 7.6 (d, J=5 Hz, 1H),7.29-7.26 (m, 1H), 7.23 (d, J=4.0 Hz, 1H), 7.05 (t, J=4.0 Hz, 1H), 6.80(d, J=9.0 Hz, 1H), 5.16 (s, 2H).

One skilled in the art will recognize that other compounds describedbelow were prepared in a similar manner to the procedures describedabove.

N1-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)-N2-methyloxalamide (231) wasprepared by substituting methanolic ammonia in Scheme 43 withmethanamine (2N solution in THF) and by substituting tert-butyl(2-amino-4-(thiophen-2-yl)phenyl)carbamate in Scheme 42 with tert-butyl(3-amino-4′-fluoro-[1,1′-biphenyl]-4-yl)carbamate. In this case, bocdeprotection was substituted by a nitro reduction usingpalladium/hydrogen as described earlier. ESI-MS: m/z 286 ([M−H]⁻), 1HNMR (400 MHz, d6-DMSO): δ 9.95 (s, 1H), 8.84 (d, J=5.2 Hz, 1H),7.56-7.53 (m, 3H), 7.29-7.18 (m, 3H), 6.84 (d, J=8.4 Hz, 1H), 5.09 (s,2H), 2.75 (s, 3H).

Synthesis ofN-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)-3-((dimethylamino)methyl)azetidine-1-carboxamide(213)

To a solution of 4′-fluoro-4-nitro-[1,1′-biphenyl]-3-amine (1.5 g, 6.46mmol) in DCM (50 mL) were added triphosgene (1.917 g, 6.46 mmol) and TEA(8.5 g, 84 mmol) at 0° C. The reaction mixture was stirred at roomtemperature for two hours. tert-butyl azetidin-3-ylmethylcarbamate(1.203 g, 6.46 mmol) was then added at 0° C. and the reaction mixturewas stirred at room temperature for four hours. The reaction crude wasdiluted with saturated citric acid solution and extracted with DCM. Theorganic layer was washed with water, brine, dried over Na₂SO₄, filteredand concentrated to yield crude residue which was purified by columnchromatography eluting with 30% EtOAc in Hexane to a fiord tert-butyl((1-((4′-fluoro-4-nitro-[1,1′-biphenyl]-3-yl)carbamoyl)azetidin-3-yl)methyl)carbamate(2 g, 69.7% yield).

To a solution of tert-butyl(1-(4′-fluoro-4-nitrobiphenyl-3-ylcarbamoyl)azetidin-3-yl)methylcarbamate(2.0 g, 4.5 mmol) in DCM (60 mL) was added TFA (8 mL, 104 mmol) at 0° C.The reaction was warmed to room temperature and stirred for 2 h. Thereaction was then concentrated under reduced pressure. The residue wasbasified with a saturated aqueous solution of NaHCO₃ and extracted witha 15% MeOH in DCM. The organic layer was dried over Na₂SO₄ andconcentrated to afford3-(aminomethyl)-N-(4′-fluoro-4-nitro-[1,1′-biphenyl]-3-yl)azetidine-1-carboxamide(1.6 g, 100% yield) as yellow syrup which is used in the next step assuch without any further purification.

To a solution of3-(aminomethyl)-N-(4′-fluoro-4-nitro-[1,1′-biphenyl]-3-yl)azetidine-1-carboxamide(2 g, 5.81 mmol) in MeOH (50 mL) were added formaldehyde (1.74 g, 58.1mmol) and acetic acid (1.74 g, 29.0 mmol) at room temperature andstirred for 1 h. NaCNBH₃ (1.46 g, 23.23 mmol) was then added at 0° C.The reaction mixture was stirred at room temperature for two hours. Thereaction was quenched with saturated NaHCO₃ and extracted with EtOAc.The organic layer was washed with water, brine and dried over Na₂SO₄.The organic layer was concentrated under vacuum to afford crude residuewhich was purified by column chromatography eluting with 2% MeOH in DCMto yield3-((dimethylamino)methyl)-N-(4′-fluoro-4-nitro-[1,1′-biphenyl]-3-yl)azetidine-1-carboxamide(1.69 g, 78% yield).

To a solution of3-((dimethylamino)methyl)-N-(4′-fluoro-4-nitro-[1,1′-biphenyl]-3-yl)azetidine-1-carboxamide(1.7 g, 4.57 mmol, 1.0 eq.) in methanol (100 mL) and was added 10% Pd/C(1.0 g, 0.94 mmol). The reaction mixture was stirred 2 h under ahydrogen atmosphere. The reaction was then filtered and the filtrate wasconcentrated under reduced pressure to giveN-(4-amino-4′-fluoro-[1,1′-biphenyl]-3-yl)-3-((dimethylamino)methyl)azetidine-1-carboxamide(35 mg, 3% yield) as an off-white solid. ESI+MS: m/z 343 ([M+H]⁺), 1HNMR(500 MHz, DMSO-d₆): δ 7.61 (s, 1H), 7.53-7.51 (m, 2H), 7.36 (d, J=2.0Hz, 1H), 7.21-7.15 (m, 3H), 6.76 (d, J=8.5 Hz, 1H), 4.98 (s, 2H), 3.99(t, J=8.0 Hz, 2H), 3.58 (dd, J_(1,2)=6.0 Hz, J_(1,3)=8.0 Hz, 2H),2.72-2.69 (m, 1H), 2.45-2.44 (m, 2H), 2.13 (s, 6H).

Example 3 Inhibition of Histone Deacetylase Enzymatic Activity

The following trypsin-coupled protocol and Caliper protocol describedherein was used to assay the compounds of the invention.

Trypsin Coupled Assay: HDAC1-KineticIC50-3 Hr PreIncubation WorkingStocks

-   -   1×HDAC Assay Buffer:    -   50 mM HEPES (GIBCO 15630-114), 100 mM KCl, 0.001% Tween-20        (Zymed 00-3005), 0.05% BSA (Invitrogen, P2489), pH 7.4    -   1.5×HDAC+1.5×Trypsin: 225 nM Trypsin(Worthinton)+0.18 μg/ml        HDAC1 (BPS Inc) in assay buffer    -   300× Trypsin stock is made up in HDAC Assay Buffer, aliquoted        and stored at −80° C.    -   3× Substrate: 18 μM Substrate in assay buffer    -   BPS Enzyme and Substrate amounts (per well)

HDAC class Enz amount [Substrate] (μM) Substrate HDAC1 Ia 3.5 ng* 6*1600*Protocol (Costar 3573 384-well plate—black/black bottom)Assay is performed at room temp. reagents are stored on ice1) Add 20 μl of enzyme+trypsin in assay buffer (Combi)2) Pin transfer compounds (100 nl pins)3) incubate 3 hours at room temp4) Add 10 ul of 3× substrate in assay buffer (Combi)5) centrifuge: 1 min at 1000 rpm6) Kinetic assay: read plates on Envision with excitation=355 nm;emission=460 nm for 7 times with interval of 10 min7) use slope from 20 min-60 min (linear range) for data analysis

HDAC2-KineticIC50-3 Hr PreIncubation Working Stocks

-   -   1×HDAC Assay Buffer:    -   50 mM HEPES (GIBCO 15630-114), 100 mM KCl, 0.001% Tween-20        (Zymed 00-3005), 0.05% BSA (Invitrogen, P2489), pH 7.4    -   1.5×HDAC+1.5× Trypsin: 225 nM Trypsin(Worthinton)+0.2 g/ml HDAC2        (BPS Inc) in assay buffer    -   300× Trypsin stock is made up in HDAC Assay Buffer, aliquoted        and stored at −80° C.    -   3× Substrate: 13.8 μM Substrate in assay buffer    -   BPS Enzyme and Substrate amounts (per well)

HDAC class Enz amount [Substrate] (μM) Substrate HDAC2 Ia 4 ng* 4.5*1600*Protocol (Costar 3573 384-well plate—black/black bottom)Assay is performed at room temp. reagents are stored on ice1) Add 20 al of enzyme+trypsin in assay buffer (Combi)2) Pin transfer compounds (100 nl pins)3) incubate 3 hours at room temp4) Add 10 al of 3× substrate in assay buffer (Combi)5) centrifuge: 1 min at 1000 rpm6) Kinetic assay: read plates on Envision with excitation=355 nm;emission=460 nm for 7 times with interval of 10 min7) use slope from 20 min-60 min (linear range) for data analysis

Protocol: HDAC3-KineticIC50-3 Hr PreIncubation Working Stocks

-   -   1×HDAC Assay Buffer:    -   50 mM HEPES (GIBCO 15630-114), 100 mM KCl, 0.001% Tween-20        (Zymed 00-3005), −0.05% BSA (Invitrogen, P2489), pH 7.4    -   1.5×HDAC: 0.1 μg/ml HDAC3 (BPS Inc) in assay buffer    -   3× Substrate+3× Trpsin: 28.5 μM Substrate+450 nM Trpsin in assay        buffer    -   300× Trypsin stock is made up in HDAC Assay Buffer, aliquoted        and stored at −80° C.    -   BPS Enzyme and Substrate amounts (per well)

HDAC class Enz amount [Substrate] (μM) Substrate HDAC3 Ia 2 ng* 9.5*1600*Protocol (Costar 3573 384-well plate—black/black bottom)Assay is performed at room temp. reagents are stored on ice1) Add 20 al of enzyme+trypsin in assay buffer (Combi)2) Pin transfer compounds (100 nl pins)3) incubate 3 hours at room temp4) Add 10 al of 3× substrate in assay buffer (Combi)5) centrifuge: 1 min at 1000 rpm6) Kinetic assay: read plates on Envision with excitation=355 nm;emission=460 nm for 7 times with interval of 10 min7) use slope from 20 min-60 min (linear range) for data analysis

The compounds of the invention were assayed for histone deacetylaseinhibitory activity. The data is presented in the tables below. The datais presented whereby the letter “A” means the compound has an IC₅₀between 0.0000001 μM≦0.1 μM, the letter “B” means the compound has anIC₅₀ between 0.11 μM≦1 μM, the letter “C” means the compound has an IC₅₀between 1.1 μM≦5 μM, the letter “D” means the compound has an IC₅₀between 5.1 μM≦30 μM, the letter “E” means the compound has an IC₅₀>30μM. It will be recognized by one skilled in the art that the compoundscan be assessed against other histone deacetylase enzymes and that thepresentation of data is illustrative and in no way intended to limit thescope of the present invention. The compounds of the invention can beassayed against a range of histone deacetylase enzymes depending uponthe performance activity desired to be gathered. Furthermore, theletters “A”, “B”, “C”, “D” and “E” are also illustrative and in no wayis intended to limit the scope of the present invention. For example,the symbol “E” is not meant to indicate that a compound necessarilylacks activity or utility but rather that its IC₅₀ value against theindicated histone deacetylase enzyme is greater than 30 μM.

TABLE 2 HDAC1 HDA2 HDAC3 HDAC1 HDAC2 HDAC3 Kinetic Kinetic KineticCaliper Caliper Caliper Cmpd IC50 IC50 IC50 IC50 IC50 IC50 No (uM) (uM)(uM) (uM) (uM) (uM) 3 B B C B B B 2 C B C 24 C B E 23 D D D 6 D C E 21 DC C 1 C B C C C C 120 C B C 4 A A C A B B 81 C B D 30 A A B A A B 27 C CD 12 B B C A A B 11 C B D 22 B B D B B C 16 A A B A A B 13 A A C A A B 5C C D 45 A A C A A C 17 A A C A A C 83 D D E 76 B B C 36 E E E 35 A A CA A C 19 B B E 46 B B D 34 C B D 78 D D E 20 B C D 47 C B D 49 A A D A BC 32 A A B A A B 40 A A C A B C 59 C C D 48 B B C 8 C D E 73 C B D B B C42 B B D 10 D D E 43 E D E 9 C C D 31 B B E 26 C C E 61 C B D 89 B B D BB C 68 A A C A A C 37 D D E 85 B B C 59 D C E 82 D D E 62 E D E 69 B B D79 C B C 80 D D E 41 D D E 33 B A D 66 B B D 15 B A C B B B 91 B A C A AB 64 C B D 38 C C C 75 D C E 57 A A C A B C 44 B B D 55 C B E 58 D C D65 B A D 51 C C D 53 B B E B B C 39 A A D A A C 72 C B D B B C 54 A A CA A B 18 A A C A A B 86 B A C 74 D D E 87 A A C 88 B A C 90 C B E 25 A AC A A B 67 B A C A A C 52 C B D 60 B B C B B C 77 D C D 63 B B C B B B70 C B D B B D 56 A A C A B C 96 A B C 97 D C C 106 A A C A A B 124 A AD A A C 128 A A D A B C 129 A A D A A C 130 B B D 98 A A C A B C 133 A AC A B C 114 A A D 109 B B E 92 A A C 107 D D C D D C 113 A A D A A D 115A A D 116 B B D 131 C C E 100 B B C 126 A A D A B D 104 D D D 95 B B D137 A A C 117 A A C 94 A B D 122 A A D 123 A A C 139 A A C A B C 105 C BD 141 A A D 110 A A D 111 A A D 134 A A D A B C 140 C B D 138 B C E 127D D E 136 C C E 135 B C E 108 C B D 132 C C E 112 C B E 119 A A D 142 BB C 145 B B C B B C 125 A A D 93 C B D 146 B B D 147 A B C 143 B A D 144A A C A B C 118 A A C A A C 121 B A B A B B 177 A A C 14 A B C 50 D C E103 A A D 152 D C E 153 D C D 154 C B C 155 C B E 156 A A C 157 B B D158 A A D 159 B B D 160 A A C 161 D C E 162 C B D 163 B B C 164 A A C165 E E E 166 B A C 167 B A B 168 B B D 169 B B C 170 B A B 171 A B C172 A A B 173 E D E 174 A A B 175 C C D 176 E D E 177 A B C 178 A B D181 C C D 182 A A C 185 A A B 186 A A D A B C 187 A A D B B E 191 B B C193 A A B 194 A A B 200 C B D 195 C C E 196 E D E 197 D D E 198 C C D199 B B E 201 E E E 202 A B C 203 A B C 204 B B C 205 B B D 206 D D C207 D D B 208 A B C 209 A B c 210 A A C 211 A B E 212 A A B 213 B C D214 A A D 215 A A C 216 A A D 217 A A C 218 A B A 219 B B B 220 E D C221 E D C 222 D D B 223 C C B 224 A B A 225 C D E 226 A A D 227 A A A228 D E C 229 C D B 230 A B A 231 C D D 232 A A D 233 A A D 234 B C A235 B B D 236 A B C 237 D E C 238 A B E 239 C C D 240 E D D 241 A B C242 A A C 243 D D B 244 B B B 245 A A C 246 B B B

Example 4 Methods

Learning tests: All behavioral testing is described in Fischer et al.,Neuron 48, 825-838 (2005).

Fear Conditioning Tests:

Context-dependent fear conditioning. Training consists of a 3 minexposure of mice to the conditioning box (context) followed by a footshock (2 sec, 0.5/0.8/1.0 mA, constant current). The memory test isperformed 24 hr later by re-exposing the mice for 3 min into theconditioning context. Freezing, defined as a lack of movement except forheart beat and respiration associated with a crouching posture, isrecorded every 10 sec by two trained observers (one is unaware of theexperimental conditions) during 3 min (a total of 18 samplingintervals). The number of observations indicating freezing obtained as amean from both observers is expressed as a percentage of the totalnumber of observations. For short term memory test, the memory test isperformed 3 hrs after the foot shock training.

Tone-dependent fear conditioning. Training consists of a 3 min exposureof mice to the conditioning box (context), followed by a tone [30 sec,20 kHz, 75 dB sound pressure level (SPL)] and a foot shock (2 sec, 0.8tnA, constant current). The memory test is performed 24 hr later byexposing the mice for 3 min to a novel context followed by an additional3 min exposure to a tone (10 kHz, 75 dB SPL). Freezing is recorded every10 sec by two nonbiased observers as described above.

Morris water maze test. The water maze paradigm is performed in acircular tank (diameter 1.8 m) filled with opaque water. A platform(11×11 cm) is submerged below the water's surface in the center of thetarget quadrant. The swimming path of the mice is recorded by a videocamera and analyzed by the Videomot 2 software (TSE). For each trainingsession, the mice are placed into the maze consecutively from fourrandom points of the tank. Mice are allowed to search for the platformfor 60 s. If the mice do not find the platform within 60 s, they aregently guided to it. Mice are allowed to remain on the platform for 15s. Two training trials are given every day; the latency for each trialis recorded for analysis. During the memory test (probe test), theplatform is removed from the tank, and the mice are allowed to swim inthe maze for 60 s.

Spatial working memory on elevated T-maze. Mice are maintained on arestricted feeding schedule at 85% of their free-feeding weight. Spatialworking memory is first assessed on an elevated plastic T-maze. Thisconsists of a start arm (47×10 cm) and two identical goal arms (35×10cm), surrounded by a 10 cm high wall. A plastic food well is located 3cm from the end of each goal arm. The maze is located 1 m above thefloor in a well lit laboratory that contained various prominent distalextramaze cues. The mice are habituated to the maze, and to drinkingsweetened, condensed milk, over several days before spatialnon-matching-to-place testing.

Each trial consists of a sample run and a choice run. On the sample run,the mice are forced either left or right by the presence of a plasticblock, according to a pseudorandom sequence (with equal numbers of leftand right turns per session, and with no more than two consecutive turnsin the same direction). A reward consisting of 0.07 ml of sweetened,condensed milk (diluted 50/50 with water) is available in the food wellat the end of the arm. The block is then removed, and the mouse isplaced, facing the experimenter, at the end of the start arm and alloweda free choice of either arm. The time interval between the sample runand the choice run is approximately 15 s. The animal is rewarded forchoosing the previously unvisited arm (that is, for alternating). Miceare run one trial at a time with an inter-trial interval (ITI) ofapproximately 10 min. Each daily session consists of 4 trials, and micereceive 24 trials in total.

Cannulation and injection: Microcannula are inserted into the lateralbrain ventricles as described by Fischer et al., J. Neurosci 22, 3700-7(2002).

Immunoblotting and staining: Lysates for immunoblotting are prepared asdescribed by Fischer et al. Neuron 48, 825-838 (2005). To isolatehistones, brain tissue is homogenized in TX-buffer (50 mM Tris HCl, 150mM NaCl, 2 mM EDTA, 1% Triton-100) and incubated at 4° C. for 15 minprior to centrifugation at 200 rpm for 10 min. After a wash-step inTX-buffer the pellet is dissolved in TX-buffer containing 0.2M HCl andincubated on ice for 30 min, before a second centrifugation at 10000 rpmfor 10 min. The supernatant is either dialysed or directly used forimmunoblotting. Antibodies are used in 1:1000 concentrations. Allantibodies detecting histones and anti-PSD-95 are from e.g., Upstate(Lake Placid, N.Y.). Anti-synaptophysin (SVP38) is from e.g., Sigma.Anti-neuronal nuclei (neuN) and anti-growth associated protein (Gap43)are from e.g., Chemicon (Temecula, Calif.) and anti-N-cadherin,anti-beta-catenin are from e.g., Santa Cruz (Santa Cruz, Calif.).Immunostaining is performed as described by Fischer et al., Neuron 48,825-838 (2005). Antibodies mentioned above are used in a 1:500 dilution.Anti-MAP-2 antibody (e.g., Sigma) is used in a 1:200 dilution.

Statistical analysis: The data are analyzed by unpaired student's t testand one-way ANOVA (ANalyis Of VAriance). One-way ANOVA followed bypost-hoc Scheffe's test is employed to compare means from severalgroups.

Example 5

Compounds of the invention are tested for their ability to reinstatementlearning behavior through inhibition of HDAC. Brain atrophy occursduring normal aging and is an early feature of neurodegenerativediseases associated with impaired learning and memory. Only recentlyhave mouse models with extensive neurodegeneration in the forebrain beenreported. One of these models is the bi-transgenic CK-p25 Tg mice whereexpression of p25, a protein implicated in various neurodegenerativediseases (Cruz, J., et al., Curr. Opin. Neurobiol. 14, 390-394 (2004)),is under the control of the CamKII promoter and can be switched on oroff with a doxycycline diet (Fisher, A. et al., Neuron 48, 471-83(2003); Cruz, J. et al., Neuron 40, 471-83 (2003)). Post-natal inductionof p25 expression for 6 weeks causes learning impairment that isaccompanied by severe synaptic and neuronal loss in the forebrain.

Specifically, compounds of the invention are tested for their ability toreinstate learning behavior and to recover access to long-term memoriesin CK-p25 Tg mice that had developed synaptic and neuronal loss. p25 isinduced in 11 month old CK-p25 Tg mice for 6 weeks. Afterwards p25expression is repressed and one group of CK-p25 Tg mice are subjected todaily or intermittent (e.g every other day) injection of a compound ofthe invention for a period of time (e.g. 1-4 weeks), whereas the othergroup receives saline injection. Subsequently all mice, including acontrol group that do not express p25, are subjected to fearconditioning and water maze learning. To this end, p25 expression isinduced in 11-month old CK-p25 mice for 6 weeks, before one group isinjected daily or intermittently (e.g every other day) for a period oftime (e.g. 1-4 weeks) with a compound of the invention whereas thecontrol group receives saline. The freezing behavior and spatiallearning of vehicle vs. compound treated groups are compared and levelsof synaptic marker proteins are measured. Freezing is defined as a lackof movement except for heart beat and respiration associated with acrouching position. Brain atrophy and hippocampal neuronal loss are alsoevaluated.

The effect of HDAC inhibition using a compound of the invention on therecovery of inaccessible long-term memories is evaluated. Eleven monthold CK-p25 Tg mice are trained in the fear conditioning paradigm andreturned to their home cages for 4 weeks. Subsequently p25 is inducedfor 6 weeks before the mice are injected with either saline (vehicle) ora compound of the invention for a period of time. Afterwards all mice,including a vehicle injected control groups that did not express p25 anda group of compound-injected control mice that were not trained, aresubjected the memory test. The freezing behavior and spatial learning ofvehicle vs. compound treated groups are compared and levels of synapticmarker proteins are measured. Brain atrophy and hippocampal neuronalloss are also evaluated. A significant reduction in freezing behaviorduring the memory test (P<0.0001) test, suggests the loss ofconsolidated long-term memories.

Example 6

Compounds of the invention are tested to determine their effect onplasticity factors in CK-p25 Tg mice that developed severeneurodegeneration. p25 is induced in 11 month old CK-p25 Tg mice for 6weeks. Afterwards p25 expression is repressed and one group of CK-p25 Tgmice is subjected to daily or intermittent (e.g every other day)compound injections for a period of time (e.g 1-4 weeks), whereas theother group receives saline injection. Hippocampal neuronal loss isevaluated for compound and vehicle treated mice for example, bycomparing images showing hippocampal NeuN and MAP-2 staining and usingimmunoblots from the hippocampus and cortex of all groups.

Example 7

Compounds of the invention are tested to determine their effect onlearning, basal anxiety, explorative behavior and brain plasticity.C57BL/6J mice are subjected to fear conditioning and are injectedintraperitoneally [ip] with a compound of the invention or salineimmediately afterwards. Freezing behavior is evaluated during a memorytest performed 24 h later.

C57BL/6J mice are implanted with microcannulae into the lateral brainventricles (icv) and are injected with either a compound of theinvention or vehicle immediately after fear conditioning. Freezingbehavior is evaluated during a memory test performed 24 h later.

C57BL/6J mice are injected [ip] daily or intermittmently with a compoundof the invention or saline for a period of time (e.g 1-4 weeks) beforeall mice are subjected to the elevated plus maze and open field test.MAP-2 immunoreactivity (IR) in the hippocampus is evaluated and compoundtreated and vehicle treated mice compared. Markers for brain plasticityare measured.

Example 8

Compound of the invention are tested to determine their ability torecover spatial memories. CK-p25 Tg mice in which p25 expression isrepressed and control mice are trained in the water maze paradigm untilall mice reliably find the hidden platform. Afterwards all mice arereturned to their home cages for 4 weeks to allow the consolidation ofhippocampus independent long-term memories. Afterwards p25 expression isinduced for 6 weeks, followed by p25 repression. One group of CK-p25 Tgmice are injected daily or intermittently (e.g every other day) with acompound of the invention, whereas the other group is injected withvehicle. Pilot studies have shown that a probe test, commonly used toanalyze memory retrieval in the water maze paradigm, is not a reliableread out to analyze long-term memory retrieval. In fact, withoutextensive re-training even wild type mice show no significant preferencefor the target quadrant when tested 10 weeks after the training in aprobe test. To measure the retrieval of long-term memory mice areinstead exposed to only 2 reminder-training sessions on a single day.The mean escape latency during the reminder-training sessions iscompared to control mice that did not receive the initial training.Escape latency is evaluated for compound treated and control mice.

Example 9

Compounds of the invention are tested to determine the specificacetylation mark elicited by the compound which is relevant to thetreatment of Rubinstein Taybi. Sagittal brain sections of RubinsteinTaybi CBP+/− mice are immunostained to detect levels of AcH2B inhippocampal neurons. Western blot analysis of hippocampal proteinextracts from CBP+/− and WT mice using antibodies against 13-actin, H2B(nonacetylated), AcH2A, AcH3 and AcH4 is used to reveal AcH2B level.Quantification of Western blot analysis shows the differences in thelevel of p-actin, total H2B, AcH2A, and AcH3.

Example 10

Compounds of the invention were evaluated in primary neuronal culturefor determining HDAC inhibitor effects in cells. E17 embryonic mouseforebrain was dissociated into a single cell suspension by gentletrituration following trypsin/DNAse digestion. Cells were plated at adensity of 12,500 cells per well in poly-D-lysine/laminin-coatedblack/clear bottom 96-well plates (BD Biosciences #BD356692) inNeurobasal medium containing 2% B27, and 1% pen/strep. Cultures weretreated with HDAC inhibitors for 24 h starting on the third day afterplating at varying inhibitor concentrations.

Compounds of the invention were evalutated to determine the functionalmeasures of compounds' cellular HDAC activity (Immunoflourescentanalysis). Imaging of neurons was performed using automated microscopy.After 24 h of HDAC inhibitor treatment, formaldehyde dissolved inphosphate-buffered saline (PBS) was added directly to the wells for afinal concentration of 4%. Cells were fixed for 10 minutes at roomtemperature. Following two washes with phosphate-buffered saline, cellswere permeabilized and blocked with blocking buffer composed of 0.1%Triton X-100, 2% BSA in PBS. For immunofluorescence imaging of histonemodifications, cells were stained with anti-acetyl-H3K9 (Millipore,cat#07-352) or anti-Ac—H4K12 (Millipore, cat#04-119) antibodies, andAlexa488-conjugated secondary antibody (Molecular Probes). Cellularnuclei were identified by staining with Hoechst 33342 (Invitrogen,H3570). Cell nuclei and histone acetylation signal intensity weredetected and measured using a laser-scanning microcytometer (Acumen eX3,TTP Laptech). Acumen Explorer software was used to identify a thresholdof histone acetylation signal intensity such that, in the absence ofHDAC inhibitor, >99.5% of cells had intensity levels below thethreshold. In the presence of HDAC inhibitors, cells with histoneacetylation signal intensities above the threshold were scored as“bright green nuclei”. The percentage of nuclei scoring as bright greenwas quantitated for each HDAC inhibitor. This percentage was thennormalized to DMSO controls. Representative images of histoneacetylation staining in neurons (with dendrites stained by anti-Map2Bantibody (EnCor) and Alexa-555-conjugated secondary antibody (MolecularProbes); shown in red) in the absence and presence of HDAC inhibitorswere taken on a Zeiss Observer Z1 microscope.

Example 11

Materials and Methods: CBP mutant mice(B6.Cg-Tg(Camk2a-Crebbp*)1364Tabe/J) are obtained from, for example, theJackson lab. Expression of this FLAG-epitope tagged, dominant negativetruncation of the CREB-binding protein (FLAG-CBPA1, lacking the codingsequence for amino acids 1084-2441) is spatially directed to neurons inthe forebrain (hippocampus, amygdala, striatum, and cortex) andtemporally directed to postnatal development by the CaMKIIa promoter.This dominant negative mutant form of CBP (designed to interrupttranscription factors utilizing CBP as a co-activator for the expressionof their target genes) is expressed from the transgene at 95% ofendogenous CBP levels in the hippocampus and 84% of endogenous CBPlevels in the cortex. Hemizygous mice exhibit hippocampus-dependentmemory deficits (such as reduced long-term potentiation, defectivespatial learning, and impaired contextual fear conditioning) with noneof the developmental impairments observed in CBP-deficient mutantmodels. The CBP mutant hemizygous mice and their control littermates areinjected daily of intermittently (e.g every other day) with vehicle or acompound of the invention for a period of time (e.g 10 days). On day 11,mice are trained in contextual fear conditioning paradigm (Trainingconsists of a 3 min exposure of mice to the conditioning box (context,TSE) followed by a foot shock (2 sec, 0.8 mA, constant current). Onehour after training, mice are injected with compounds of the inventionor vehicle. On day 12 mice are returned to the training box and thefreezing behavior were monitored and recorded. References: Learn Mem.2005 March-April; 12(2): 111-9. Transgenic mice expressing a truncatedform of CREB-binding protein (CBP) exhibit deficits in hippocampalsynaptic plasticity and memory storage. Wood M A, Kaplan M P, Park A,Blanchard E J, Oliveira A M, Lombardi T L, Abel T.

CBP (+/−) heterozygous mice represent a model of the human diseasesyndrome of Rubinstien Taybi. It is the same genetic mutation which isbelieved to be casual in humans. People affected by this syndrome havememory/cognition and developmental deficits. Using the dosing paradigm(e.g., 1 mg/kg, QD, 10 days) compounds of the invention are tested fortheir ability to restore the memory of these mice to an equivalent levelas found in the wild-type littermates.

Example 12

Materials and Methods: CK/p25 mouse is an inducible neurodegenerativedisease mouse model. The bi-transgenic mice are created by crossing theCamK2a-tTA and the tetO-p25 Tg mouse lines. In the presence ofdoxycyclin, the expression of p25 is suppressed. When doxycyclin isremoved, the expression of p25 is strongly induced in the forebrain. Sixweeks of p25 induction causes massive neuronal loss, elevatedbeta-amyloid peptide production, tau associated pathology, andimpairment in learning and memory. For these experiments, doxycyclin isremoved from 3-month old CK/p25 mice and the control littermates for 6weeks. The mice are subsequently injected with compounds of theinvention or vehicle for a period of time (e.g. 10 days). After thattime period (e.g., on day 11), mice are trained in contextual fearconditioning paradigm (training consisted of a 3 min exposure of mice tothe conditioning box (context, TSE) followed by a foot shock (2 sec, 0.8mA, constant current). One hour after training, mice are injected with acompound of the invention or vehicle. On day 12 mice are returned to thetraining box and the freezing behavior is monitored and recorded.References: Cruz J C, et al. Neuron 2003, 40:471-483; Fischer A, et al,Neuron, 2005, 48: 825-838; Fischer A, et al., Nature 2007, 447: 178-182.

The p25 vehicle group represents non-induced vehicle treated mice, thep25/CK vehicle group represents the induced untreated mice. The controlgroup consists of the tetO-p25 Tg mice fed on normal diet, which do notexpress p25. The compounds of the invention are tested for their abilityto restore contextual fear conditioning learning. The brain pathology ofp25 is reminiscent of human patients with neurodegeneration and memoryimpairment.

Example 13

Mice are trained using contextual fear conditioning paradigm on day 0.For example, training consists of a 3 min exposure of mice to theconditioning box (context, TSE) followed by three foot shocks (2 sec,0.8 mA, constant current with 15-seconds-intervals). From day 1, miceare trained in extinction trials. For each training day, mice are twiceexposed to the conditioning box for 3 min without foot shock (twoextinction trials/day). One hour after the first trial, mice areinjected with a compound of the invention (e.g., 30 mg/1 (g, i.p.). Onehour after the injection, the second extinction trial is performed. Thefreezing time in each individual trial is measured.

Example 14

For the memory reconsolidation paradigm, after the fear extinctiontraining as described above, mice are re-housed in the home cage for onemonth. Mice are subsequently re-expose to the conditioning box for 3min, their freezing behavior is measured. It is well established thatafter the fear memory extinction trials, fear memory will spontaneouslyrecover after resting in the home cage for a period of time. Thespeculated mechanism for fear extinction is to trigger the formation ofnew memory which competes with fear memory and in turn reduces the fearresponse. Conversely, reconsolidation based fear memory extinctionparadigm has been proposed to directly modify the activated fear memory,so that the reduced fear response does not spontaneously recover(Extinction-reconsolidation boundaries: key to per attenuation of fearmemories. Monfils M H, Cowansage K K, Klann E, LeDoux J E. Science. 2009May 15; 324(5929):951-5. Epub 2009 Apr. 2).

Compounds of the invention are tested in several dosing paradigms interms of frequency and dose in multiple disease states. The experimentsabove can show treatment is beneficial for extinction of aversivememories. In an embodiment, the compounds of the present invention inconjunction with proper training paradigm lead to permanent erasing offear memory.

Example 15 Caliper Endpoint Assay

The following non-trypsin coupled in-vitro HDAC enzymatic endpoint assaywas used to assay the compounds of the invention. Below is astandardized protocol for running HDAC selectivity panel on CaliperLabChip EZ-Reader Instrument.

The Caliper HDAC Assay Buffer (acronym HAB, 1 liter) was prepared asfollows:

Components: Final Concentration: Catalog #s: 100 mL 1M KCL 100 mM Sigma#9541-500 G 50 mL 1M HEPES, 50 mM Sigma #H3375-1 KG pH 7.4 1 mL 10% BSA0.01% *(important) SeraCare #AP-4510-80-100 G 20 uL 50% 0.001% Zymed#00-3005-20 mL Tween-20

The buffer was added to 1 liter Milli-Q water and store at 4° C. * BSAfinal concentration cannot exceed 0.01% for use on the Caliperinstrument. HDAC enzymes 1 to 9 were purchased from BPS Bioscience (seetable below for catalog #s).

The substrate (stock conc.) was prepared as follows:

Substrate A (aka HS-C2 (structure shown below): 10 mM/2 mM in 100%DMSO)—final conc. 2 μM-HDACs 1, 2, 3, 6Substrate B (aka HS-CF3 (structure shown below): 10 mM/2 mM in 100%DMSO)—final conc. 2 μM-HDACs 4, 5, 7, 8, 9

The quench inhibitor (stock conc.) was LBH (structure shown below). Theinstrument buffer was ProfilerPro Separation Buffer (e.g., Caliper#760367). The instrument chip was LabChip EZ Reader II 12-SipperOff-Chip Mobility Shift Chip (e.g., Caliper #760404).

The Caliper peptide structures HS-C2 and HS-CF3 are shown below andprepared according to the synthetic procedure described in U.S. patentapplication Ser. No. 61/628,562 entitled “Fluorescent Substrates forDetermining Lysine Deacetylase Activity” filed Nov. 2, 2011.

The protocol was carried out as follows:

-   -   1. Caliper LabChip and 1 μM Marker (peptide in separation        buffer) were prepared for instrument run.    -   2. Warm up Caliper HAB buffer to room temperature    -   3. Pin 100 nl compd. into 20 μl 1.5× solution HDACs and        preincubate 3 hrs at room temperature    -   4. Add 10 μl 3× solution acetylated substrate to initiate the        reaction for 50 minutes.    -   5. Stop reaction with 5 μL of 10 μM LBH solution (˜1.4 uM final)    -   6. Mix plate    -   7. Read plate on EZ Reader instrument. Separate substrate and        product peaks by capillary electrophoresis and read fluorescence        from both substrate and product.    -   8. Run parameters were as follows:

Post sample Upstream Downstream buffer Final Peak Pressure votage votagesip time delay order HS-C2 −1.3 −500 −1500 35 90 Product first HS-CF3−1.3 −500 −1500 35 90 Product firstBelow is the HDAC and Peptide concentration table.

Peptide Stock Final Conc. enz. enz. Conversion HDAC BPS Cat. # Peptide(μM) (μM) (nM) % @ 1 hr 1 50051 C2 2 4.82 5 27% 2 50002 C2 2 44 3 20% 2T(#X083-59 C2 2 19.1 0.5 XTAL sol'n 0.9 mg/ml) 3 50003 C2 2 7.67 5 30% 450004 CF3 2 26.6 0.5 38% 5 50045 CF3 2 0.567 1 17% 6 50006 C2 2 5.66 229% 7 50007 CF3 2 8.97 0.5 45% 8 50008 CF3 2 12.93 0.5 22% 9 50009 CF3 257.99 3 25%

Preparation of Substrates A and B:

In one aspect, substrates A and B were prepared as follows. To asolution of (S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanoic acid(1w) in THF was added methyl 2-aminoacetate hydrochloride (2w), Et₃N andHATU. The mixture was stirred at room temperature for 16 h. The reactionwas filtered through Celite. The reaction filtrate was diluted with 100mL of water and stirred for 15 min. The suspension was filtered off,rinsed with water and dried to afford (S)-methyl2-(2-((tert-butoxycarbonyl)amino)-4-methylpentanamido)acetate (3w).

To a solution of (S)-methyl2-(2-((tert-butoxycarbonyl)amino)-4-methylpentanamido) acetate (3w) in1,4-dioxane was added a 5M solution of HCl in 1,4-dioxane at roomtemperature. The reaction was stirred at room temperature for 16 h. Thereaction mixture was filtered to afford (S)-methyl2-(2-amino-4-methylpentanamido)acetate hydrochloride (4w) as thefiltered solid.

To a solution of (S)-methyl 2-(2-amino-4-methylpentanamido)acetatehydrochloride (4w) in THF was added 6-((tert-butoxycarbonyl)amino)hexanoic acid, HATU and DIPEA. The reaction was stirred at roomtemperature for 18 h. The mixture was then filtered through Celite. Thefiltrate was concentrated under reduced pressure and the crude residuewas purified by column chromatography (silica gel, CH₂C12/MeOH=50/1) togive (S)-methyl13-isobutyl-2,2-dimethyl-4,11,14-trioxo-3-oxa-5,12,15-triazaheptadecan-17-oate(6w) as a white solid.

To a solution of (S)-methyl13-isobutyl-2,2-dimethyl-4,11,14-trioxo-3-oxa-5,12,15-triazaheptadecan-17-oate(6w) in THF was added a solution of LiOH.H₂O in water at roomtemperature. After 3 h, the reaction mixture was concentrated, dilutedwith water and acidified with a 1N aqueous solution of HCl to about pH4-5. The mixture was stirred for 15 min and the white precipitate formedwas filtered off, rinsed with water, and dried to afford(S)-13-isobutyl-2,2-dimethyl-4,11,14-trioxo-3-oxa-5,12,15-triazaheptadecan-17-oicacid (7w).

To a solution of 8w in DMF at room temperature was added7-amino-4-methyl-2H-chromen-2-one, HATU and triethylamine. The reactionwas stirred at room temperature for 2 h. A saturated solution of sodiumbicarbonate was added. The product was extracted with ethyl acetate. Thecombined organic layers were washed with water, dried over sodiumsulfate, filtered and concentrated under reduced pressure. The cruderesidue was purified by prep-HPLC to afford 9w.

To a solution of 9w in 1,4-dioxane was added a 5M solution of HCl in1,4-dioxane. The reaction was stirred at room temperature for 3 h. Themixture was concentrated under reduced pressure to afford 10w.

To a solution of 10w in THF was added 7, HATU and triethylamine. Thereaction was stirred at room temperature for 3 h. A saturated solutionof sodium bicarbonate was added. The product was extracted with ethylacetate. The combined organic layers were washed with water, dried oversodium sulfate, filtered and concentrated under reduced pressure. Thecrude material was purified by silica gel column (prep-HPLC) to afford11w.

To a solution of 11w in 1,4-dioxane and was added a 5M solution of HClin 1,4-dioxane. The reaction was stirred at room temperature for 3 h.The reaction was then concentrated and dried under reduced pressure toafford 12w.

To a solution of 12w in THF at room temperature was added 13w, BOP andtriethylamine. The reaction was stirred at room temperature for 22 h.The mixture was then filtered through Celite. The filtrate wasconcentrated under reduced pressure. The crude residue was purified byprep-HPLC to give 14w.

Example 16 Histone Acetylation Changes in Neuronal Cell Culture afterTreatment with Compounds of the Invention

Primary cultured mouse forebrain neurons were treated for 24 hours withcompounds of the invention, and the resulting increases in histoneacetylation were measured with immunofluorescence assays. FIGS. 1A, 1B,1C and 1D show dose response curves for induction of acetylation ofhistone H4 lysine 12, and histone H3 lysine 9 for compounds of theinvention. Specifically, FIG. 1A and FIG. 1C are dose response curves ofhistone acetylation (AcH4K12) in primary neuronal cultures by compoundsof the invention. FIG. 1B and FIG. 1D are dose response curves ofhistone acetylation (AcH3K9) in primary neuronal cultures by compoundsof the invention. For FIGS. 1A, 1B, 1C, and 1D, the cultures weretreated for 24 hours with compounds of the invention at the indicateddoses and histone acetylation was measured in an immunofluorescenceassay. The percentage of cells with strong histone acetylation staining(bright green cells) was quantitated and averaged across duplicatesamples from two independent cultures.

The protocol for primary neuronal culture was carried out as follows.E17 embryonic mouse forebrain was dissociated into a single cellsuspension by gentle trituration following trypsin/DNAse digestion.Cells were plated into poly-D-lysine/laminin coated black/clear bottom96-well plates (e.g., BD Biosciences #BD356692), 6-well plates, or 10 cmplates in Neurobasal medium containing 2% B27, and 1% pen/strep.Cultures were treated with compounds of the invention for 24 h startingon the thirteenth day after plating at varying concentrations of thecompounds of the invention.

The protocol for histone acetylation immunofluorescence assays usingprimary neuronal culture was carried out as follows. Imaging of neuronswas performed using automated microscopy. After 24 h of treatment bycompounds of the invention, formaldehyde dissolved in phosphate-bufferedsaline (PBS) was added directly to the wells for a final concentrationof 4%. Cells were fixed for 10 minutes at room temperature. Followingtwo washes with phosphate-buffered saline, cells were permeabilized andblocked with blocking buffer composed of 0.1% Triton X-100, 2% BSA inPBS. For immunofluorescence imaging of histone modifications, cells werestained with anti-acetyl-H3K9 (e.g., Millipore, cat#07-352) oranti-Ac—H4K12 (e.g., Millipore, cat#04-119) antibodies, andAlexa488-conjugated secondary antibody (e.g., Molecular Probes).Cellular nuclei were identified by staining with Hoechst 33342 (e.g.,Invitrogen, H3570). Cell nuclei and histone acetylation signal intensitywere detected and measured using a laser-scanning microcytometer (e.g.,Acumen eX3, TTP Laptech). Acumen Explorer software was used to identifya threshold of histone acetylation signal intensity such that, in theabsence of compounds of the invention, >99.5% of cells had intensitylevels below the threshold. In the presence of the compounds of theinvention, cells with histone acetylation signal intensities above thethreshold were scored as “bright green nuclei”. The percentage of nucleiscoring as bright green was quantitated for compounds of the invention.This percentage was then normalized to DMSO controls.

Example 17 Use of Mass Spectrometry for Identification of Changes inHistone Acetylation and Methylation States Induced in Neurons byTreatment with Compounds of the Invention

The protocol was carried out as follows. Large-scale primary neuronalcultures were treated with compound 103 (at 1 and 10 μM) for 24 hours,and cells were harvested for mass spectrometry analysis of histoneacetylation and methylation according to methods developed by S. L.Peach et al. (see Quantitative assessment of “ChIP-grade” antbodiesdirected against histone modifications reveals patterns of co-occurringmarks on histone protein molecules; Molecular & Cellular Proteomics,published on Mar. 21, 2012 as manuscript M111.015941). FIG. 2 is a heatmap indicating changes in histone acetylation and methylation statesregulated by treatment of primary neuronal cultures with compound 103 ofthe invention or compound A (positive control). Specifically, FIG. 2 isa heat map indicating the mass spectrometry characterization of histoneacetylation and methylation states regulated treatment of neuronalcultures with compounds of the invention. Primary neuronal cultures weretreated for 24 hours at the indicated doses. Arrows indicate the histonemarks states induced equipotently by control compound A and compound103.

Changes in many histone marks were induced in a dose-dependent manner.However, in the case of three specific histone mark states (AcH3K27/di-or tri-MeH3K36; triMeH3K27/triMeH3K36), compound A and compound 103 wereequipotent. Thus, these three sets of histone marks may representspecific states that respond selectively to inhibition of HDACs 1/2.

Primary Neuronal Culture:

E17 embryonic mouse forebrain was dissociated into a single cellsuspension by gentle trituration following trypsin/DNAse digestion.Cells were plated into poly-D-lysine/laminin coated black/clear bottom96-well plates (e.g., BD Biosciences #BD356692), 6-well plates, or 10 cmplates in Neurobasal medium containing 2% B27, and 1% pen/strep.Cultures were treated with compounds of the invention for 24 h startingon the thirteenth day after plating at varying concentrations of thecompounds of the invention.

Mass Spectrometry Determination of Histone Acetylation and MethylationStates:

Primary neuronal cultures grown in 10 cm plates (3 million cells/plate)were treated for 24 hours with compound 103 (1 μM and 10 μM). Cells wereharvested for proteomic analysis of histone acetylation and methylationstates. Nuclei were purified from harvested cells, and histones werecollected by acid extraction. Mass spectrometric analysis was used toidentify the indicated histone acetylation and methylation states (PeachS L, et al.).

Example 18 Identification of Gene Expression Changes Upon Treatment withCompounds of the Invention in Neurons

Changes in histone acetylation and methylation are key events in theregulation of gene transcription (Bannister A J, et al. Cell Res.21:381-95). Thus, HDAC inhibitors that alter histone mark states incells also induce changes in gene expression (e.g. Chang J, et al. Br JCancer. 2012 Jan. 3; 106(1):116-25). Evidence suggests that HDACisoforms have unique roles in controlling the expression of neuronalgenes; some genes appear to be regulated by the coordinated actions ofmultiple HDACs; other genes seem to be uniquely regulated by specificHDACs (Guan J S, et al. Nature. 2009 May 7; 459(7243):55-60).

Genome-wide transcript profile studies were performed to assess thecellular activity of compounds of the invention, downstream from theirimmediate effects on histone acetylation and methylation, and todiscover gene expression signatures that were specific phenotypes ofHDAC1/2 inhibition. For these assays, primary cultured neurons weretreated with compound A or compound 215 (at 10 μM) for 24 hours, andthen harvested RNA for transcript profile analysis on Illuminamicroarrays.

1130 genes were found to be up or down-regulated by at least 1.5 fold(p<0.05; one-way ANOVA test) by compound 215. Of these, 109 wereregulated to approximately the same extent by compound A and compound215 (fold changes equivalent within +/−10%). Table 3 lists afunctionally well-annotated subset of these 109 genes, and indicates thedirectionality and fold changes induced by compound A and compound 215.These 109 genes were regulated by compound 215, which is a selectiveHDAC1,2 inhibitor.

This functionally annotated set consists of 34 genes that were upregulated, and 43 genes that were down-regulated, by compound A andcompound 215. These gene expression phenotypes may represent a signatureof selective inhibition of HDACs 1/2.

TABLE 3 Genes regulated by compound 215 in neuronal cell cultures. FoldChange Fold Change Gene compound 215 compound A Cdr2 1.9 2.1 Rgs4 2.02.0 Eno2 1.8 1.9 Tpst2 1.8 1.7 Rcl1 1.7 1.7 Ramp3 1.7 1.7 Nes 1.6 1.6Nsf 1.5 1.6 Abca7 1.6 1.6 Tmem86a 1.5 1.6 Ina 1.6 1.6 Il11ra1 1.5 1.6Urod 1.6 1.6 Fchsd1 1.5 1.6 Smyd3 1.5 1.6 St8sia5 1.4 1.5 Tmem184b 1.51.5 Actn4 1.4 1.5 Wipi1 1.5 1.5 Arsa 1.5 1.5 Snhg11 1.4 1.5 Pou3f1 1.51.5 Galnt2 1.4 1.5 Med10 1.5 1.5 Adam15 1.4 1.5 Ddn 1.4 1.5 Eif4ebp1 1.71.5 Dok5 1.5 1.5 Bbc3 1.6 1.5 Lmtk2 1.6 1.5 Adrbk2 1.5 1.5 Cry2 1.5 1.5Reep6 1.5 1.4 Zfyve27 1.5 1.4 Rap2c −1.5 −1.4 Scn2a1 −1.5 −1.4 Rundc3b−1.5 −1.4 Marcksl1 −1.6 −1.4 Idh1 −1.5 −1.4 Pccb −1.5 −1.5 Sltm −1.5−1.5 Dcx −1.5 −1.5 Lass2 −1.4 −1.5 Aqp4 −1.5 −1.5 Polr3k −1.4 −1.5Fam131a −1.5 −1.5 Hist1h2an −1.5 −1.5 Gabrg2 −1.6 −1.5 Pgam5 −1.5 −1.5Hist1h2ad −1.7 −1.5 Ccdc53 −1.5 −1.5 Suv420h1 −1.6 −1.5 Hist1h2ai −1.6−1.5 Efhal −1.5 −1.5 Fbxo6 −1.5 −1.5 Mgat3 −1.4 −1.6 Dctn6 −1.4 −1.6Lsm5 −1.4 −1.6 Chchd1 −1.5 −1.6 Astn1 −1.4 −1.6 Npm3-ps1 −1.5 −1.6 Dis31−1.5 −1.6 Mrpl48 −1.5 −1.6 Smarca1 −1.6 −1.6 Gpr22 −1.7 −1.6 Aldoc −1.5−1.6 Hist1h2af −1.6 −1.6 Hmgn2 −1.7 −1.7 Cenpa −1.7 −1.7 Taf9b −1.7 −1.8Jazf1 −1.6 −1.8 Mrpl54 −1.7 −1.8 Ppp1r9a −1.8 −1.8 Nasp −1.7 −1.9 Pbk−1.7 −1.9 Lor −2.1 −2.0 Nxph2 −1.9 −2.1

The protocol for genome-wide transcript profiling assays was carried outas follows. E17 embryonic mouse forebrain was dissociated into a singlecell suspension by gentle trituration following trypsin/DNAse digestion.Primary neuronal cultures grown in 6-well plates were treated for 24hours with compound 215 at 10 μM. RNA was isolated with the RNeasy kit(e.g., Qiagen) according to the manufacturer's instructions. cDNA wassynthesized using ArrayScript (e.g., Ambion). Triplicate samples werecollected and analyzed using Illumina mouse whole-genome-6 microarrays.Total RNA from the samples was normalized to 20 ng/l, and the Illumina®TotalPrep™-96 RNA Amplification Kit (e.g., Applied Biosystems, PN#4393543) protocol was used for amplification in a semi automatedprocess. The total RNA underwent reverse transcription to synthesizefirst-strand cDNA. This cDNA was then converted into a double-strandedDNA template for transcription. In vitro transcription synthesized aRNAand incorporated a biotin-conjugated nucleotide. The aRNA was thenpurified to remove unincorporated NTPs, salts, enzymes, and inorganicphosphate. Labeled cRNA was normalized to 150 ng/μl and hybridized toIllumina's MouseWG-6 v2.0 Expression BeadChip. The labeled RNA strandwas hybridized to the bead on the BeadChip containing the complementarygene-specific sequence. After 16 hours of hybridization, the beadchipswere washed and stained using a Cy3 streptavidin conjugate. Illumina'sBeadArray Reader was used to measure the fluorescence intensity at eachaddressed bead location. Raw data were annotated with Genome Studio(Illumina), and then quantile normalized and baseline transformed to themedian of the DMSO control samples using GeneSpring software (Agilent).Probes failing to score as “present” in 100% of samples from at leastone treatment condition were removed. A statistical threshold (p<0.05,paired t-test with Benjamini-Hochberg multiple comparisons correction)and a fold change criterion (≧1.5) were then applied to generate listsof genes up- and down-regulated by compound 215.

Example 19 Biochemical Profiling of Compounds of the Invention UsingNative HDAC2 Complex Enzymatic Assays

In cells, HDACs1/2 are assembled into large multi-protein complexes.Three major complexes containing HDACs 1/2 have been found to bind tothe non-selective hydroxamic acid HDAC inhibitor SAHA: CoREST, NuRD, andSIN3 (Bantscheff M, et al. Nat Biotechnol. 2011 March; 29(3):255-65).

The ability of compounds of the invention to selectively inhibitHDACs1/2 to effect unique conformational changes in the enzymes inducedby assembly into these multi-protein complexes, which are not achievedby the recombinant free enzymes tested in the in vitro assays, wasevaluated. HDAC2 complexes from mouse forebrain were immunoprecipitated.The following determinations were made: 1) known complex members(CoREST, mSin3a, and Mta3), but not HDAC3, were present in the HDAC2immunoprecipitates (FIG. 3A); 2) the immunoprecipitated HDAC2 complexeswere enzymatically active, as confirmed by one of our standard in vitroassays (FIG. 3B); 3) the effects of compound A, compound 215, and anegative control compound on this enzymatic activity. FIG. 3C shows thatboth compound A and compound 215 inhibited the enzymatic activity of theHDAC2 immunoprecipitate, whereas the negative control compound producedno inhibition, indicating that HDAC2 is sensitive to inhibition bycompounds of the invention in the context of its endogenousmulti-protein complexes.

FIG. 3A, FIG. 3B, and FIG. 3C indicate that HDAC2 complexesimmunoprecipitated from mouse forebrain are enzymatically active andsensitive to inhibition by compounds of the invention. FIG. 3A is aWestern blot showing detection of HDACs 1, 2 and 3 and known members ofendogenous HDAC2 complexes in immunoprecipitated HDAC2 complexes. FIG.3B is a bar chart indicating the enzymatic activity of theimmunoprecipitated HDAC2 complexes from mouse forebrain.Immunoprecipitates were added to an in vitro enzymatic assay containingMAZ1600 substrate, incubated for 22 hours prior to fluorescencedetection of the activity. FIG. 3C is a graph indicating the %inhibition of enzymatic activity over time by compounds of the inventionor compound A (positive control). The HDAC2 immunoprecipitates wereincubated for three hours in the presence of the indicated compounds andthen added to the MAZ1600 in vitro enzymatic assay for three hours ofincubation prior to fluorescence detection.

Example 20 Learning and Memory—In Vivo Biomarker Analysis and EfficacyStudies in Normal Mice and a Mouse Model of Neurodegeneration, CK-p25Mice

Compound 54 and compound 103 were examined for their efficacy in themodel behavior paradigm, the contextual fear conditioning task asdescribed in Example 4. It was found that daily injections of eithercompound 54 or compound 103 (10 mg/kg daily i.p.) enhanced associativelearning in wildtype mice (see FIG. 4). This experiment was repeated inan inducible mouse model of neurodegeneration, the CK-p25 mouse (seeFIG. 5). This mouse model recapitulates many of the hallmarks ofAlzheimer's disease (e.g. profound neuronal loss in the forebrain,increased β-amyloid peptide production, tau hyperphosphorylation, andsevere cognitive impairment). Following the treatment of CK-p25 micewith either compound 54 (10 mg/kg daily i.p.) or compound 103 (1 mg/kgdaily i.p.), it was observed that these compounds were able toameliorate the cognitive deficits compared to vehicle treatment.

FIG. 4 is a bar chart indicating increased freezing in a contextual fearconditioning paradigm following administration of compounds of theinvention or compound A (positive control; 10 mg/kg) in wild type mice.Administration of compound 54 (10 mg/kg), compound 103 (10 mg/kg), orcompound A (positive control; 10 mg/kg) for ten days prior to fearconditioning training enhances associative learning in wild type mice(n=10 per group). *P<0.05, **P<0.01, ***P<0.001, using Tukey's testafter ANOVA.

FIG. 5 is a bar chart indicating increased freezing in a contextual fearconditioning paradigm following administration of compounds of theinvention or compound A (positive control; 1 mg/kg) in 6-week inducedCK-p25 mice (neurodegenerative mouse model). Administration of compound54 (10 mg/kg), compound 103 (1 mg/kg), or compound A (positive control;1 mg/kg) enhances associative learning in 6-week induced CK-p25mice(numbers in histogram columns=animal n's). *P<0.05, **P<0.01,***P<0.001, using Tukey's test after ANOVA.

After completing the behavioral tests, the mice were sacrificed andtheir brain tissue was subjected to either immunohistochemistry orimmunoblotting. Immunoblotting experiments demonstrated that compound 54administration marginally increased global histone acetylation in thehippocampus (see FIG. 6A, FIG. 6B, and FIG. 7). In addition, compound103 administration at 1 mg/kg substantially decreased immunoreactivityfor glial fibrillary acidic protein (GFAP) in the brains of treatedanimals, indicating reduced astrogliosis and inflammation (see FIG. 8Aand FIG. 8B).

FIG. 6A and FIG. 6B indicate that chronic administration (10 days) ofcompound 54 (10 mg/kg) and compound 103 (10 mg/kg) increases theacetylation of histone loci H3K9 and/or H4K12 in the cortex of normalmice. Compound A is the positive control. FIG. 6A is a bar chartindicating the relative histone acetylation of H3K9 and H4K12 in C57BL/6mouse cortex following chronic administration of compounds of theinvention or compound A (positive control) compared to untreated(vehicle) mice. FIG. 6B shows the representative Western blots.

FIG. 7 is an immunoblot analysis of histone acetylation (AcH4K5 andAcH4K12) in CK-p25 mouse hippocampus following the chronicadministration of compounds of the invention or compound A (positivecontrol). Chronic administration of compound 54 (10 mg/kg) marginallyincreases acetylation of histone residues, H4K5 and H4K12, in CK-p25mice (n=3 per group).

FIGS. 8A and 8B indicate that the administration of compound 103 (1mg/kg) significantly decreases GFAP protein levels, an astrocytic markercommonly associated with brain inflammation (n=2). FIG. 8A is a Westernblot showing the decrease of GFAP protein after administration ofcompound 103 of the invention. FIG. 8B is a bar chart indicating thelevel of GFAP normalized to GAPDH. *P<0.05, **P<0.01, using Student ttest.

Specifically, the above results directed to acetylation in mouse cortexwere obtained by a 2-step Crude Protein Lysis protocol, which wasadapted from ‘Lysis of Mammalian Cells and Tissue in Gel-LoadingBuffer,’ from Molecular Cloning: A Laboratory Manual, Book #3, Chapter18 (Detection and Analysis of Proteins Expressed from Cloned Genes),pages 62-63.

For whole frozen mouse brain, the follow procedures were carried out:

1. On ice, thaw frozen tissue and dissect whole cortex using majorneuroanatomical landmarks.

-   -   a. Immediately homogenize carefully in 250 uL of ice-cold        suspension buffer. 100 uL was used for tissue approx. 2-3 mm³        and was adjust as needed. 1.5 mL disposable pestles (e.g.,        Fisher cat#03-392-100) were used.    -   b. As soon as possible, add an equal volume of 2×SDS gel-loading        buffer, pipetting up and down to mix.        2. Place the sample at 95° C. for 5 min.        3. Shear viscous chromosomal DNA by smoothly passaging through        23-25 gauge hypodermic needle (2-3×) or by sonicating briefly        (the needle method also worked fine). Avoid foaming/bubbles.        4. Centrifuge the sample at 10,000 g for 10 min at room        temperature, transferring supernatant to fresh tube.        5. Aliquot sample as needed based on protein concentration.

The suspension buffer was prepared using 0.1M NaCl, 0.01M TrisCl (pH7.6), and 0.001M EDTA (pH 8.0). The buffer could have be prepared aheadand stored at room temperature. Just before use, the following wasadded: 1× phosphatase/protease inhibitor cocktail (e.g., ThermoFisher“HALT,” cat#78440), and 5 mM Sodium Butyrate (HDAC inhibitor).

The 2×SDS gel-loading buffer was prepared using 100 mM TrisCl (pH 6.8),4% SDS, and 20% glycerol. The buffer could have been prepared ahead andstored at room temperature. Just before use, the following was added:200 mM dithiothreitol (from 1M stock) and 5 mM Sodium Butyrate (HDACinhibitor).

Further, the Western blots above were obtained as follows. 10 ugcortical extract per sample on BioRad 15% Tris-HCl Gel at 150V wasresolved for 1 hour. The sample was transfer to PVDF at 350 mA for 1hour. The membranes were blocked with TBST+5% milk for 1 hour, thenincubated overnight with primary antibody against i) H3K9ac (e.g.,Millipore #07-352); ii) H3-C terminus (e.g., Millipore #07-108); iii)H4K12ac (e.g., Millipore #04-119); or iv) Histone H4 (H4pan, e.g.,Millipore #07-108). The membranes were subsequently incubated withsecondary HRP-linked antibodies against i) Mouse IgG (e.g., CellSignaling #7076) or ii) Rabbit IgG (e.g., Cell Signaling #7074) for 1hour. Chemiluminescent signal was generated using Supersignal West DuraExtended Duration Substrate (ThermoFisher #34076) and radiographic film.Quantification was executed using Image J software (e.g., NIH).

Example 21 In Vivo Efficacy in Mood Related Behaviors

The compounds of the invention were tested in various behavioralparadigms. The compounds of the invention were prepared at the beginningof each experiment (10-12 aliquots per experiment) in aliquots. Allaliquots were frozen and only thawed one time for administration. Eachaliquot contained the appropriate concentration of compounds of theinvention in 100% DMSO. Prior to administration, aliquots were removed,thawed, and prepared as:

45% PEG400/45% saline (0.9% NaCl)/10% DMSO: 200 ul DMSO, 900 ul PEG400,900 ul Saline 30% Cremophor EL/65% Saline/5% DMSO: 100 ul DMSO, 600 ulCremophor, 1300 ul Saline. Aliquots were discarded each day and freshcompound was prepared each day. About 2 mls of compound for each day foreach drug (2 ml is the amount used for the calculations below) weremade.

Administration of compounds of the invention was carried out as follows.Each mouse was administered compounds of the invention daily atapproximately the same time each day. Mice were weighed daily as a grossmeasure of overall tolerability of the compounds of the invention. Micewere administered the appropriate dose of the compound of the inventionat either 5 mls or 10 mls/kg, depending on the solubility of thecompound the invention. Mice were returned to the home cage immediatelyafter for the first 5 days of dosing. For the rest of dosing, mice wererun in various behavioral paradigms (detailed below) and administeredcompounds of the invention after the completion of the behavior runs.Thus, all behavioral data were obtained 18-24 hours after the previousdrug administration. All behavior data should reflect the chroniceffects of the compounds of the invention and not acute effects of eachof the compounds of the invention.

The behavioral assays were prepared as follows. For theamphetamine-induced hyperactivity (AIH) assay, mice were habituated onday 1 (day 6 of compound administration), baseline motor activity wasobtained on day 2 (day 7 of compound administration), and the effects ofthe compounds of the invention on the amphetamine response were obtainedon day 3 (day 8 of compound administration).

Specifically, AIH was examined in eight identical open-field chambers(16.5″×16″×12″; AccuScan Instruments, Columbus, Ohio). See FIGS. 9A, 9B,10A and 10B. Activity was detected by infrared beam breaks and recordedautomatically by VersaMax software (AccuScan). Daily sessions wereautomatically binned in 5 minute intervals (VersaDat; AccuSacn) forstatistical analysis. AIH was run over three consecutive days.

FIG. 9A and FIG. 9B indicate that chronic administration (8 days) ofcompound 103 (30 mg/kg) in normal mice attenuates the response inamphetamine induced hyperactivity. FIG. 9A is a line graph of the totallocomotor activity over time (min) in C57BL/6 mice after chronicadministration of compound 103 of the invention in the amphetamineinduced hyperactivity (AIH) mouse model. FIG. 9B is a bar chart of thetotal activity during the test period.

FIG. 10A and FIG. 10B indicate that the chronic administration (8 days)of compound 191 (10 mg/kg) in normal mice attenuates the response inamphetamine induced hyperactivity. FIG. 10A is a graph of the totallocomotor activity over time (min) in C57BL/6 mice following chronicadministration of compound 191 of the invention in AIH. FIG. 10B is abar chart of the total activity during the test period. Decreasedlocomotor activity is indicative of an antimanic phenotype.

For the forced-swim test (FST), mice were exposed to the 6 minute teston day 9 of compound administration (See FIG. 11 and FIG. 12).Specifically, mice were placed in one of five identical cylindricalchambers (24 cm×15 cm) filled˜½ way with warm water (26±+2). The FST wasrun in one day for 6 minutes and behavior was scored automatically(EthoVision; Noldus) for the final 4 minutes of the session.

FIG. 11 is a bar chart indicating that chronic administration ofcompound 191 in C57BL/6 mice decreases the immobility time during theforced swim test. Decreased immobility is indicative of anantidepressant phenotype.

FIG. 12 is a bar chart indicating that chronic administration ofcompound 54 in C57BL/6 mice decreases the immobility time during theforced swim test. Decreased immobility is indicative of anantidepressant phenotype.

Further, tissue collection and biochemistry analysis were carried out onday 10. Mice were treated with compounds of the invention and tissue wascollected one hour after the final administration. Mice were sacrificedvia cervical dislocation and the brains were rapidly removed andimmediately flash frozen on dry ice. Cortex and striatum were thendissected from whole brain and tissue was prepared and analyzed.Specific details of tissue collection, dissection and Western Blottingcan be found in Example 20. (see 2-step Crude Protein Lysis protocol anddetails on Western blots).

1. A compound having formula I:

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof, wherein: U is selected from single bond,CR^(2e)R^(2f)—CR^(2g)R^(2h), NR^(2d), NR^(2d)—NR^(2d′), and O; J isselected from NH₂, OH, and SH; V is selected from C and N, provided thatwhen V is N, one of R^(2a), R^(2b), or R^(2c) is absent; each X isindependently selected from hydrogen, deuterium, methyl, CF₃, andhalogen; R^(2a) is selected from hydrogen, halogen, OH, NH₂, and C₁-C₈alkyl; R^(2b) is selected from hydrogen, halogen, OH, NH₂, and C₁-C₈alkyl; R^(2c) is selected from hydrogen, halogen, OH, NH₂, and C₁-C₈alkyl; R^(2d) is selected from NH₂, hydrogen, and C₁-C₈ alkyl; R^(2d′)is selected from NH₂, hydrogen, and C₁-C₈ alkyl; R^(2e), R^(2f), R^(2g),and R^(2h) are each independently selected from hydrogen, halogen, andC₁-C₄ alkyl; or taken together two of R^(2a), R^(2b), and R^(2c) form═O, or taken together two of R^(2a), R^(2b), and R^(2c) form a C₃-C₈cycloalkyl ring, C₄-C₈ cycloalkenyl ring, or 3 to 8 membered saturatedor partially unsaturated heterocyclic ring and the remaining R^(2a),R^(2b), or R^(2c) is absent or selected from hydrogen, halogen, OH, NH₂,and C₁-C₈ alkyl, further wherein said cycloalkyl, cycloalkenyl, andheterocyclic ring are unsubstituted or substituted with one or moreR^(x); or alternatively, taken together two of R^(2a), R^(2b), R^(2c)and R^(2d) form a C₃-C₈ cycloalkyl ring, C₄-C₈ cycloalkenyl ring, or 3to 8 membered saturated or partially unsaturated heterocyclic ring andthe remaining R^(2a), R^(2b), or R^(2c) is selected from hydrogen,halogen, OH, NH₂, and C₁-C₈ alkyl or the remaining R^(2d) is hydrogen,NH₂, or C₁-C₈ alkyl, further wherein said cycloalkyl, cycloalkenyl, andheterocyclic ring are unsubstituted or substituted with one or moreR^(x); or taken together two of R^(2a), R^(2b), and R^(2c) form anaromatic or heteroaromatic ring and the remaining R^(2a), R^(2b), orR^(2c) is absent, provided that when two of R^(2a), R^(2b), and R^(2c)form an aromatic ring and the remaining R^(2a), R^(2b), or R^(2c) isabsent, U is not a single bond when t is 0, further wherein saidaromatic and heteroaromatic ring are unsubstituted or substituted withone or more R^(x), or taken together R^(2e) and R^(2f) or R^(2g) andR^(2h) taken together form ═O; or taken together two of R^(2e), R^(2f),R^(2g), and R^(2h) on two adjacent carbon atoms together with the bondbetween said adjacent carbon atoms form a carbon-carbon double bond; ortaken together two of R^(2e), R^(2f), R^(2g), and R^(2h) on two adjacentcarbon atoms together with the intervening atoms to which they areattached form a 3 to 8 membered saturated or partially saturated ring;each R^(x) is independently selected from (CH₂)_(z)NH₂, (CH₂)_(z)NHR³,(CH₂)_(z)NR³R³, OR³, OCF₃, OCH₂F, OCHF₂, (CH₂)_(z)-aromatic ring,(CH₂)_(z)-heterocyclic ring, hydroxyl, halogen, C₁-C₈ alkyl, (C₁-C₈alkyl)CF₃, (C₁-C₈ alkyl)OH, C(O)R^(3′)(CH₂)_(z)C(O)NH₂,(CH₂)_(z)C(O)NHR³, (CH₂)_(z)C(O)NR³R³, (CH₂)_(z)NHC(O)R⁴, and(CH₂)_(z)NR⁴C(O)R⁴. or taken together two R^(x) attached to the samecarbon atom of a cycloalkyl, cycloalkenyl or heterocyclic ring togetherform ═O; or taken together two R^(x) form a C₃-C₈ cycloalkyl ring, C₄-C₈cycloalkenyl ring or 3 to 8 membered saturated or partially unsaturatedheterocyclic ring, further wherein said cycloalkyl, cycloalkenyl, andheterocyclic ring are unsubstituted or substituted with one or moreR^(z); or taken together two R^(x) form an aromatic ring orheteroaromatic ring, further wherein said aromatic and heteroaromaticring are unsubstituted or substituted with one or more R^(z); each R^(z)is independently selected from halogen, C₁-C₄ alkyl, OH, OR³, CF₃, OCF₃,OCH₂F, OCHF₂, NH₂, NHR³, NR³R³, and C(O)CH₃; each R³ is independentlyselected from C₁-C₈ alkyl and O(C₁-C₈ alkyl); each R⁴ is independentlyselected from C₁-C₈ alkyl and CF₃; R⁵ is selected from OR⁶ wherein R⁶ isC₂-C₈ alkyl, C₂-C₈ alkyl, C₂-C₈ alkenyl, (CH₂)_(u)-5-6 memberedsaturated, unsaturated, or partially unsaturated heterocyclic ring,(CH₂)_(v)—C₄-C₈ cycloalkyl ring, (C₁-C₈-alkyl)_(w)-C₄-C₈cycloalkenylring, (CH₂)_(s)-aromatic ring, and (CH₂)_(s)-heteroaromatic, and whereinsaid heterocyclic, cycloalkyl, cycloalkenyl, heteroaromatic, andaromatic ring are unsubstituted or substituted with one or more R^(y)and said alkenyl is substituted with one or more R^(T); each R^(y) isindependently selected from halogen, OR⁶, NH₂, NHR⁶, NR⁶R⁶, OH, CF₃,aromatic ring, C(O)R⁶, C₁-C₈ alkyl, C₂-C₈ alkenyl, and C₂-C₈ alkynyl;each R⁶ is an independently selected C₁-C₈ alkyl; each R^(T) isindependently selected from hydrogen, halogen, Si(R³)₃, phenyl, andC₁-C₈ alkyl; u is selected from 0, 1, and 2; v is selected from 0, 1,and 2; w is selected from 0, 1, and 2; s is selected from 0, 1, and 2; tis selected from 0, 1, and 2, and each z is independently selected from0, 1, 2, and
 3. 2. The compound of claim 1, having a formula selectedfrom:

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof, wherein U is selected from single bond andCR^(2e)R^(2f)—CR^(2g)R^(2h).
 3. The compound of claim 1 or apharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof,wherein the moiety:


4. The compound of claim 1 or a pharmaceutically acceptable salt,hydrate, solvate, or prodrug thereof, wherein R⁵ is selected from:

wherein m is selected from 2, 3, and 4;

wherein n is selected from 1, 2, 3, and 4;

wherein each Y is independently selected from CH, CR^(y), and N; and jis selected from 0, 1, 2, 3, 4, and 5;

wherein k is selected from 0, 1, 2, or 3; and

wherein each Z is independently selected from CH₂, CHR^(y), CR^(y)R^(y),O, NH, NR^(y), and S; and m is selected from 0, 1, and
 2. 5. Thecompound of claim 4 or a pharmaceutically acceptable salt, hydrate,solvate, or prodrug thereof, wherein R⁵ is selected from phenyl,2-pyridinyl, 3-pyridinyl, 4-pyridinyl, 2-pyrimidinyl, 4-pyrimidinyl,5-pyrimidinyl, 2-pyrazinyl, oxazolyl, thiazolyl, and isoxazolyl.
 6. Thecompound of claim 1 or a pharmaceutically acceptable salt, hydrate,solvate, or prodrug thereof, wherein V is C, and taken together R^(2a)and R^(2b) are:

wherein: J is selected from N, O, C, and S; when J is N or C, R^(u) isselected from hydrogen, C₁-C₈ alkyl, (C₁-C₈)CF₃, (C₁-C₈)OH, andC(O)R^(3a); and when J is O or S, R^(u) is absent; R^(3a) is C₁-C₈alkyl; v is selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8; and d and d′are each independently selected from 0, 1, 2, and
 3. 7. The compound ofclaim 6 or a pharmaceutically acceptable salt, hydrate, solvate, orprodrug thereof, wherein V is C, and taken together R^(2a) and R^(2b)form: (1) a 6-membered saturated heterocyclic ring selected from:

(2) a 5-membered saturated heterocyclic ring selected from:

(3) a 4-membered saturated heterocyclic ring selected from:

(4) a ring selected from:

wherein o and o′ are each independently selected from 0, 1, and 2; or(5) a partially unsaturated bicyclic ring selected from:

wherein o and o′ are each independently selected from 0, 1, 2, and 3; or(6) a saturated bicyclic ring selected from:

wherein o and o′ are each independently selected from 0, 1, 2, and 3;wherein: J is selected from N, O, C, and S; when J is N or C, R^(u) isselected from hydrogen, C₁-C₈ alkyl, (C₁-C₈)CF₃, (C₁-C₈)OH, andC(O)R^(3a); and when J is O or S, R^(u) is absent; R^(3a) is C₁-C₈alkyl; v is selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8; v′ is selectedfrom 0, 1, 2, 3, and 4; r and r′ are each independently selected from 0,1, and 2; and q is selected from 0, 1, 2, 3, 4, 5, 6, 7, and
 8. 8. Thecompound of claim 1 or a pharmaceutically acceptable salt, hydrate,solvate, or prodrug thereof, wherein V is C, and R^(2a)R^(2b)R^(2c)C is

wherein x is selected from 1, 2, 3, and 4; or

wherein w is selected from 1, 2, and
 3. 9. The compound of claim 1 or apharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof,wherein V is C, and taken together two of R^(2a), R^(2b), and R^(2c)form a ring selected from:

wherein: J is selected from N, O, C, and S; when J is N or C, R^(u) isselected from hydrogen, C₁-C₈ alkyl, (C₁-C₈)CF₃, (C₁-C₈)OH, C(O)R^(3a);and when J is O or S, R^(u) is absent; R^(3a) is C₁-C₈ alkyl; n is 1, 2,or 3; and v is selected from 0, 1, 2, 3, 4, 5, 6, 7, and
 8. 10. Thecompound of claim 1 or a pharmaceutically acceptable salt, hydrate,solvate, or prodrug thereof, wherein R^(2a)R^(2b)R^(2c)V(CH₂)_(t)U isselected from:

wherein: b is selected from 0, 1, 2, and 3; and a is selected from 0, 1,2, 3, 4, 5, 6, 7, 8, 9, and 10;


11. The compound of claim 1, selected from: Cmpd No structure 1

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or a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof.
 12. A pharmaceutical composition comprising an effective amountof a compound of claim 1 or a pharmaceutically acceptable salt, hydrate,solvate, or prodrug thereof and a pharmaceutical carrier, diluent, orexcipient.
 13. A method of treating, alleviating, and/or preventing acondition wherein said condition is associated with histone deacetylaseactivity in a subject comprising administering to the subject in needthereof an effective amount of a compound of claim 1 or apharmaceutically acceptable salt, hydrate, solvate or prodrug thereof.14. The method of claim 13, wherein the condition is selected from aneurological disorder, memory loss or impairment, cognitive functiondisorder or impairment, extinction learning disorder, fungal disease orinfection, inflammatory disease, hematological disease, and neoplasticdisease.
 15. The method of claim 14, wherein the condition is selectedfrom: a cognitive function disorder or impairment associated withAlzheimer's disease, Huntington's disease, seizure induced memory loss,schizophrenia, Rubinstein Taybi syndrome, Rett Syndrome, Fragile X, Lewybody dementia, vascular dementia, ADHD, dyslexia, bipolar disorder andsocial, cognitive and learning disorders associated with autism,traumatic head injury, or attention deficit disorder, anxiety disorder,conditioned fear response, panic disorder, obsessive compulsivedisorder, posttraumatic stress disorder, phobia, social anxietydisorder, substance dependence recovery or Age Associated MemoryImpairment (AAMI), or Age Related Cognitive Decline (ARCD); ahematological disease selected from acute myeloid leukemia, acutepromyelocytic leukemia, acute lymphoblastic leukemia, chronicmyelogenous leukemia, myelodysplastic syndromes, and sickle cell anemia;a cancer; and an extinction learning disorder selected from a fearextinction deficit and post-traumatic stress disorder.
 16. The method ofclaim 13, further comprising (1) administering to the subject aneffective amount of a pharmaceutically active ingredient other than acompound of claim 1, and/or (2) exposing the subject to cognitivebehavioral therapy (CBT), psychotherapy, behavioral exposure treatments,virtual reality exposure (VRE) and/or cognitive remediation therapy. 17.The method of claim 13, wherein the compound or a pharmaceuticallyacceptable salt, hydrate, solvate, or prodrug thereof is administered bya route selected from oral, parenteral, intramuscular, intranasal,sublingual, intratracheal, inhalation, ocular, vaginal, rectal, andintracerebroventricular.
 18. A method of improving memory in a normalsubject comprising administering to the subject in need thereof aneffective amount of a compound of claim 1 or a pharmaceuticallyacceptable salt, hydrate, solvate, or prodrug thereof.
 19. A method ofincreasing synaptic density, synaptic plasticity, or dendritic densityin neurons in a subject comprising administering to the subject in needof such increase an effective amount of a compound of claim 1 or apharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof.20. A method of synthesizing a compound of claim 1 or a pharmaceuticallyacceptable salt, hydrate, solvate, or prodrug thereof.
 21. A kitcontaining one or more compounds of claim 1 or a pharmaceuticallyacceptable salt, hydrate, solvate, or prodrug thereof.